# Applied Statistics for Bioinformatics using R

Wim P. Krijnen
November 10, 2009
ii
Preface
The purpose of this book is to give an introduction into statistics in order
to solve some problems of bioinformatics. Statistics provides procedures to
explore and visualize data as well as to test biological hypotheses. The book
intends to be introductory in explaining and programming elementary statis-
tical concepts, thereby bridging the gap between high school levels and the
specialized statistical literature. After studying this book readers have a suf-
ﬁcient background for Bioconductor Case Studies (Hahne et al., 2008) and
Bioinformatics and Computational Biology Solutions Using R and Biocon-
ductor (Genteman et al., 2005). The theory is kept minimal and is always
illustrated by several examples with data from research in bioinformatics.
Prerequisites to follow the stream of reasoning is limited to basic high-school
knowledge about functions. It may, however, help to have some knowledge
of gene expressions values (Pevsner, 2003) or statistics (Bain & Engelhardt,
1992; Ewens & Grant, 2005; Rosner, 2000; Samuels & Witmer, 2003), and
elementary programming. To support self-study a suﬃcient amount of chal-
lenging exercises are given together with an appendix with answers.
The programming language R is becoming increasingly important because
it is not only very ﬂexible in reading, manipulating, and writing data, but
all its outcomes are directly available as objects for further programming.
R is a rapidly growing language making basic as well as advanced statisti-
cal programming easy. From an educational point of view, R provides the
possibility to combine the learning of statistical concepts by mathematics,
programming, and visualization. The plots and tables produced by R can
readily be used in typewriting systems such as Emacs, L
A
T
E
X, or Word.
Chapter 1 gives a brief introduction into basic functionalities of R. Chap-
ter 2 starts with univariate data visualization and the most important de-
scriptive statistics. Chapter 3 gives commonly used discrete and continuous
distributions to model events and the probability by which these occur. These
distributions are applied in Chapter 4 to statistically test hypotheses from
bioinformatics. For each test the statistics involved are brieﬂy explained and
its application is illustrated by examples. In Chapter 5 linear models are ex-
plained and applied to testing for diﬀerences between groups. It gives a basic
approach. In Chapter 6 the three phases of analysis of microarray data (pre-
processing, analysis, post processing) are brieﬂy introduced and illustrated
by many examples bringing ideas together with R scrips and interpretation of
results. Chapter 7 starts with an intuitive approach into Euclidian distance
iii
and explains how it can be used in two well-known types of cluster analysis to
ﬁnd groups of genes. It also explains how principal components analysis can
be used to explore a large data matrix for the direction of largest variation.
Chapter 8 shows how gene expressions can be used to predict the diagnosis
of patients. Three such prediction methods are illustrated and compared.
gives various examples of computing important quantities such as alignment
scores. Chapter 10 introduces the concept of a probability transition matrix
which is applied to the estimation of phylogenetic trees and (Hidden) Markov
Models.
R commands come after its prompt >, except when commands are part
of the ongoing text. Input and output of R will be given in verbatim
typewriting style. To save space sometimes not all of the original output
from R is printed. The end of an example is indicated by the box . In
its Portable Document Format (PDF)
1
there are many links to the Index,
to copy and paste scripts from the PDF into the R system in order to study
its outcome. Apart from using the book to study application of statistics in
bioinformatics, it can also be useful for statistical programming.
I would like to thank my colleges Joop Bouman, Sven Warris and Jan
Peter Nap for their useful remarks on parts of an earlier draft. Many thanks
also go to my students for asking questions that gave hints to improve clarity.
Remarks to further improve the text are appreciated.
Wim P. Krijnen
Hanze University
Institute for Life Science and Technology
Zernikeplein 11
9747 AS Groningen
The Netherlands
w.p.krijnen@pl.hanze.nl
Groningen
October 2009
1
c This document falls under the GNU Free Document Licence and may be used freely
for educational purposes.
iv
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii
1 Brief Introduction into Using R 1
1.1 Getting R Started on your PC . . . . . . . . . . . . . . . . . . 1
1.2 Getting help . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Calculating with R . . . . . . . . . . . . . . . . . . . . . . . . 4
1.4 Generating a sequence and a factor . . . . . . . . . . . . . . . 4
1.5 Computing on a data vector . . . . . . . . . . . . . . . . . . . 5
1.6 Constructing a data matrix . . . . . . . . . . . . . . . . . . . 6
1.7 Computing on a data matrix . . . . . . . . . . . . . . . . . . . 8
1.8 Application to the Golub (1999) data . . . . . . . . . . . . . . 10
1.9 Running scripts . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.10 Overview and concluding remarks . . . . . . . . . . . . . . . . 13
1.11 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2 Data Display and Descriptive Statistics 17
2.1 Univariate data display . . . . . . . . . . . . . . . . . . . . . . 17
2.1.1 Pie and Frequency table . . . . . . . . . . . . . . . . . 17
2.1.2 Plotting data . . . . . . . . . . . . . . . . . . . . . . . 18
2.1.3 Histogram . . . . . . . . . . . . . . . . . . . . . . . . . 19
2.1.4 Boxplot . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.1.5 Quantile-Quantile plot . . . . . . . . . . . . . . . . . . 22
2.2 Descriptive statistics . . . . . . . . . . . . . . . . . . . . . . . 24
2.2.1 Measures of central tendency . . . . . . . . . . . . . . 24
2.2.2 Measures of spread . . . . . . . . . . . . . . . . . . . . 25
2.3 Overview and concluding remarks . . . . . . . . . . . . . . . . 26
2.4 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
v
vi CONTENTS
3 Important Distributions 31
3.1 Discrete distributions . . . . . . . . . . . . . . . . . . . . . . . 31
3.1.1 Binomial distribution . . . . . . . . . . . . . . . . . . . 31
3.2 Continuous distributions . . . . . . . . . . . . . . . . . . . . . 34
3.2.1 Normal distribution . . . . . . . . . . . . . . . . . . . . 35
3.2.2 Chi-squared distribution . . . . . . . . . . . . . . . . . 37
3.2.3 T-Distribution . . . . . . . . . . . . . . . . . . . . . . . 39
3.2.4 F-Distribution . . . . . . . . . . . . . . . . . . . . . . . 40
3.2.5 Plotting a density function . . . . . . . . . . . . . . . . 41
3.3 Overview and concluding remarks . . . . . . . . . . . . . . . . 42
3.4 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4 Estimation and Inference 47
4.1 Statistical hypothesis testing . . . . . . . . . . . . . . . . . . . 47
4.1.1 The Z-test . . . . . . . . . . . . . . . . . . . . . . . . . 48
4.1.2 One Sample t-Test . . . . . . . . . . . . . . . . . . . . 51
4.1.3 Two-sample t-test with unequal variances . . . . . . . 54
4.1.4 Two sample t-test with equal variances . . . . . . . . . 56
4.1.5 F-test on equal variances . . . . . . . . . . . . . . . . . 57
4.1.6 Binomial test . . . . . . . . . . . . . . . . . . . . . . . 58
4.1.7 Chi-squared test . . . . . . . . . . . . . . . . . . . . . 59
4.1.8 Normality tests . . . . . . . . . . . . . . . . . . . . . . 63
4.1.9 Outliers test . . . . . . . . . . . . . . . . . . . . . . . . 64
4.1.10 Wilcoxon rank test . . . . . . . . . . . . . . . . . . . . 65
4.2 Application of tests to a whole set gene expression data . . . . 66
4.3 Overview and concluding remarks . . . . . . . . . . . . . . . . 68
4.4 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
5 Linear Models 73
5.1 Deﬁnition of linear models . . . . . . . . . . . . . . . . . . . . 74
5.2 One-way analysis of variance . . . . . . . . . . . . . . . . . . . 77
5.3 Two-way analysis of variance . . . . . . . . . . . . . . . . . . 83
5.4 Checking assumptions . . . . . . . . . . . . . . . . . . . . . . 85
5.5 Robust tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.6 Overview and concluding remarks . . . . . . . . . . . . . . . . 88
5.7 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
CONTENTS vii
6 Micro Array Analysis 91
6.1 Probe data . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.2 Preprocessing methods . . . . . . . . . . . . . . . . . . . . . . 94
6.3 Gene ﬁltering . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
6.4 Applications of linear models . . . . . . . . . . . . . . . . . . 100
6.5 Searching an annotation package . . . . . . . . . . . . . . . . 104
6.6 Using annotation to search literature . . . . . . . . . . . . . . 106
6.7 Searching GO numbers and evidence . . . . . . . . . . . . . . 107
6.8 GO parents and children . . . . . . . . . . . . . . . . . . . . . 108
6.9 Gene ﬁltering by a biological term . . . . . . . . . . . . . . . . 109
6.10 Signiﬁcance per chromosome . . . . . . . . . . . . . . . . . . . 110
6.11 Overview and concluding remarks . . . . . . . . . . . . . . . . 112
6.12 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
7 Cluster Analysis and Trees 117
7.1 Distance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
7.2 Two types of Cluster Analysis . . . . . . . . . . . . . . . . . . 121
7.2.1 Single Linkage . . . . . . . . . . . . . . . . . . . . . . . 121
7.2.2 k-means . . . . . . . . . . . . . . . . . . . . . . . . . . 125
7.3 The correlation coeﬃcient . . . . . . . . . . . . . . . . . . . . 130
7.4 Principal Components Analysis . . . . . . . . . . . . . . . . . 133
7.5 Overview and concluding remarks . . . . . . . . . . . . . . . . 141
7.6 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
8 Classiﬁcation Methods 145
8.1 Classiﬁcation of microRNA . . . . . . . . . . . . . . . . . . . . 146
8.2 ROC types of curves . . . . . . . . . . . . . . . . . . . . . . . 147
8.3 Classiﬁcation trees . . . . . . . . . . . . . . . . . . . . . . . . 150
8.4 Support Vector Machine . . . . . . . . . . . . . . . . . . . . . 160
8.5 Neural Networks . . . . . . . . . . . . . . . . . . . . . . . . . 162
8.6 Generalized Linear Models . . . . . . . . . . . . . . . . . . . . 164
8.7 Overview and concluding remarks . . . . . . . . . . . . . . . . 167
8.8 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
9 Analyzing Sequences 173
9.1 Using a query language . . . . . . . . . . . . . . . . . . . . . . 173
9.2 Getting information on downloaded sequences . . . . . . . . . 174
9.3 Computations on sequences . . . . . . . . . . . . . . . . . . . 176
viii CONTENTS
9.4 Matching patterns . . . . . . . . . . . . . . . . . . . . . . . . 181
9.5 Pairwise alignments . . . . . . . . . . . . . . . . . . . . . . . . 182
9.6 Overview and concluding remarks . . . . . . . . . . . . . . . . 189
9.7 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
10 Markov Models 193
10.1 Random sampling . . . . . . . . . . . . . . . . . . . . . . . . . 193
10.2 Probability transition matrix . . . . . . . . . . . . . . . . . . . 194
10.3 Properties of the transition matrix . . . . . . . . . . . . . . . 199
10.4 Stationary distribution . . . . . . . . . . . . . . . . . . . . . . 201
10.5 Phylogenetic distance . . . . . . . . . . . . . . . . . . . . . . . 203
10.6 Hidden Markov Models . . . . . . . . . . . . . . . . . . . . . . 209
10.7 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213
10.8 Overview and concluding remarks . . . . . . . . . . . . . . . . 214
10.9 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
B References 257
List of Figures
2.1 Plot of gene expression values of CCND3 Cyclin D3. . . . . . . 20
2.2 Stripchart of gene expression values of CCND3 Cyclin D3 for
ALL and AML patients. . . . . . . . . . . . . . . . . . . . . . 20
2.3 Histogram of ALL expression values of gene CCND3 Cyclin D3. 21
2.4 Boxplot of ALL and AML expression values of gene CCND3
Cyclin D3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.5 Q-Q plot of ALL gene expression values of CCND3 Cyclin D3. 23
2.6 Boxplot with arrows and explaining text. . . . . . . . . . . . 29
3.1 Binomial probabilities with n = 22 and p = 0.7 . . . . . . . . 34
3.2 Binomial cumulative probabilities with n = 22 and p = 0.7. . . 34
3.3 Graph of normal density with mean 1.9 and standard deviation
0.5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.4 Graph of normal distribution with mean 1.9 and standard de-
viation 0.5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.5 χ
2
5
-density. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.6 χ
2
5
distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.7 Density of T
10
distribution. . . . . . . . . . . . . . . . . . . . . 39
3.8 Distribution function of T
10
. . . . . . . . . . . . . . . . . . . . 39
3.9 Density of F
26,10
. . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.10 Distribution of F
26,10
. . . . . . . . . . . . . . . . . . . . . . . . 41
4.1 Acceptance and rejection regions of the Z-test. . . . . . . . . . 50
4.2 Acceptance and rejection regions of the T
5
-test. . . . . . . . . 52
4.3 Rejection region of χ
2
3
-test. . . . . . . . . . . . . . . . . . . . . 59
5.1 Plot of SKI-like oncogene expressions for three patient groups. 81
5.2 Plot of Ets2 expression values for three patient groups. . . . . 81
ix
x LIST OF FIGURES
6.1 Mat plot of intensity values for a probe of MLL.B. . . . . . . . 93
6.2 Density of MLL.B data. . . . . . . . . . . . . . . . . . . . . . . 93
6.3 Boxplot of the ALL1/AF4 patients. . . . . . . . . . . . . . . . 97
6.4 Boxplot of the ALL1/AF4 patients after median subtraction
and MAD division. . . . . . . . . . . . . . . . . . . . . . . . . 97
6.5 Venn diagram of seleced ALL genes. . . . . . . . . . . . . . . . 100
6.6 Boxplot of the ALL1/AF4 patients after median subtraction
and MAD division. . . . . . . . . . . . . . . . . . . . . . . . . 100
7.1 Plot of ﬁve points to be clustered. . . . . . . . . . . . . . . . . 122
7.2 Tree of single linkage cluster analysis. . . . . . . . . . . . . . . 122
7.3 Example of three without clusters. . . . . . . . . . . . . . . . 123
7.4 Three clusters with diﬀerent standard deviations. . . . . . . . 123
7.5 Plot of gene ”CCND3 Cyclin D3” and ”Zyxin” expressions for
ALL and AML patients. . . . . . . . . . . . . . . . . . . . . . 124
7.6 Single linkage cluster diagram from gene ”CCND3 Cyclin D3”
and ”Zyxin” expressions values. . . . . . . . . . . . . . . . . 124
7.7 K-means cluster analysis. . . . . . . . . . . . . . . . . . . . . . 126
7.8 Tree of single linkage cluster analysis. . . . . . . . . . . . . . . 126
7.9 Plot of kmeans (stars) cluster analysis on CCND3 Cyclin D3
and Zyxin discriminating between ALL (red) and AML (black)
patients. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
7.10 Vectors of linear combinations. . . . . . . . . . . . . . . . . . . 135
7.11 First principal component with projections of data. . . . . . . 135
7.12 Scatter plot of selected genes with row labels on the ﬁrst two
principal components. . . . . . . . . . . . . . . . . . . . . . . 138
7.13 Single linkage cluster diagram of selected gene expression values.138
7.14 Biplot of selected genes from the golub data. . . . . . . . . . . 144
8.1 ROC plot for expression values of CCND3 Cyclin D3. . . . . . 149
8.2 ROC plot for expression values of gene Gdf5. . . . . . . . . . 149
8.3 Boxplot of expression values of gene a for each leukemia class. 151
8.4 Classiﬁcation tree for gene for three classes of leukemia. . . . . 151
8.5 Boxplot of expression values of gene a for each leukemia class. 154
8.6 Classiﬁcation tree of expression values from gene A, B, and C
for the classiﬁcation of ALL1, ALL2, and AML patients. . . . 154
8.7 Boxplot of expression values from gene CCND3 Cyclin D3 for
ALL and AML patients . . . . . . . . . . . . . . . . . . . . . 156
LIST OF FIGURES xi
8.8 Classiﬁcation tree of expression values from gene CCND3 Cy-
clin D3 for classiﬁcation of ALL and AML patients. . . . . . 156
8.9 rpart on ALL B-cel 123 data. . . . . . . . . . . . . . . . . . . 159
8.10 Variable importance plot on ALL B-cell 123 data. . . . . . . 159
8.11 Logit ﬁt to the CCND3 Cyclin D3 expression values. . . . . . 171
9.1 G + C fraction of sequence ”AF517525.CCND3” along a win-
dow of length 50 nt. . . . . . . . . . . . . . . . . . . . . . . . 178
9.2 Frequency plot of amino acids from accession number AF517525.CCND3.179
9.3 Frequency plot of amino acids from accession number AL160163.CCND3.179
10.1 Graph of probability transition matrix . . . . . . . . . . . . . 196
10.2 Evaluation of models by AIC . . . . . . . . . . . . . . . . . . . 216
10.3 Tree according to GTR model. . . . . . . . . . . . . . . . . . . 217
xii LIST OF FIGURES
List of Tables
2.1 A frequency table and its pie of Zyxin gene. . . . . . . . . . . 18
3.1 Discrete density and distribution function values of S
3
, with
p = 0.6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2 Built-in-functions for random variables used in this chapter. . 42
3.3 Density, mean, and variance of distributions used in this chapter. 43
7.1 Data set for principal components analysis. . . . . . . . . . . 134
8.1 Frequencies empirical p-values lower than or equal to 0.01. . . 146
8.2 Ordered expression values of gene CCND3 Cyclin D3, index
2 indicates ALL, 1 indicates AML, cutoﬀ points, number of
false positives, false positive rate, number of true positives,
true positive rate. . . . . . . . . . . . . . . . . . . . . . . . . 170
9.1 BLOSUM50 matrix. . . . . . . . . . . . . . . . . . . . . . . . 186
xiii
xiv LIST OF TABLES
Chapter 1
Brief Introduction into Using R
To get started a gentle introduction to the statistical programming language
R will be given (R Development Core Team, 2009), speciﬁc for our purposes.
This will solve the practical issues to follow the stream of reasoning. In
particular, it is brieﬂy explained how to install R and Bioconductor, how to
obtain help, and how to perform simple calculations.
Since many computations are essentially performed on data vectors, sev-
eral basic illustrations of this are given. With respect to gene expressions the
data vectors are placed one beneath the other to form a data matrix with
the genes as rows and the patients as columns. The idea of a data matrix is
extensively explained and illustrated by several examples. A larger example
consists of the classical Golub et al. (1999) data, which will be analyzed
frequently to illustrate statistical procedures.
1.1 Getting R Started on your PC
your favorite operating system (Windows, Linux or MacOS) and simply follow
the instructions. After a little patience you should be able to start R (Ihaka
& Gentleman, 1996) after which a screen is opened with the prompt >. The
input and output of R will be displayed in verbatim typewriting style.
All useful functions of R are contained in libraries which are called ”pack-
ages”. The standard installation of R makes basic packages available such
as base and stats. From the button Packages at cran.r-project.org it
can be seen that R has a huge number of packages available for a wide scale
1
2 CHAPTER 1. BRIEF INTRODUCTION INTO USING R
of statistical procedures. To download a speciﬁc package you can use the
following.
> install.packages(c("TeachingDemos"),repo="http://cran.r-project.org",
+ dep=TRUE)
This installs the package TeachingDemos developed by Greg Snow from the
repository http://cran.r-project.org. By setting the option dep to TRUE
the packages on which the TeachingDemos depend are also installed. This is
strongly recommended! Alternatively, in the Windows application of R you
can simply click on the Packages button at the top of your screen and follow
the instructions. After installing you have to load the package in order to use
its functions. For instance, to produce a nice plot of the outcome of throwing
twelve times with a die, you can use the following.
> library(TeachingDemos)
> plot(dice(12,1))
In the sequel we shall often use packages from Bioconductor, a very useful
open source software project for the analysis and comprehension of genomic
data. To follow the book it is essential to install Bioconductor on your PC
or network. Bioconductor is primarily based on R and can be installed, as
follows.
> source("http://www.bioconductor.org/biocLite.R")
> biocLite()
it, and to make the ALL data (Chiaretti, et. al, 2004) available for usage, you
can use the following.
> biocLite("ALL")
> library(ALL)
> data(ALL)
These data will be analyzed extensively later-on in Chapter 5 and 6. General
help on loaded Bioconductor packages becomes available by openVignette().
For further information the reader is referred to www.bioconductor.org or
to several other URL’s
1
.
1
http://mccammon.ucsd.edu/
~
bgrant/bio3d/user_guide/user_guide.html
http://rafalab.jhsph.edu/software.html
http://dir.gmane.org/gmane.science.biology.informatics.conductor
1.2. GETTING HELP 3
In this and the following chapters we will illustrate many statistical ideas
by the Golub et al. (1999) data, see also Section 1.8. The golub data become
available by the following.
2
> library(multtest)
> data(golub)
R is object-oriented in the sense that everything consists of objects belonging
to certain classes. Type class(golub) to obtain the class of the object golub
and str(golub) to obtain its structure or content. Type objects() or ls()
to view the currently loaded objects, a list probably growing soon to be large.
To prevent conﬂicting deﬁnitions, it is wise to remove them all at the end of
a session by rm(list=ls()). To quit a session, type q(), or simply click on
the cross in the upper right corner of your screen.
1.2 Getting help
All functionalities of R are well-organized in so-called packages. Use the func-
tion library() to see which packages are currently installed on your oper-
ating system. The packages stats and base are automatically installed, be-
cause these contain many basic functionalities. To obtain an overview of the
content of a package use ls(package:stats) or library(help="stats").
Help on the purpose of speciﬁc functions can be obtained from the (package)
manual by typing a question mark in front of a function. For instance, ?sum
gives details on summation. In case you are seeking help on a function which
uses if, simply type apropos("if"). When you are starting with a new con-
cept such as ”boxplot”, it is convenient to have an example showing output
(a plot) and programming code. Such is given by example(boxplot). The
function history can be useful for collecting previously given commands.
Type help.start() to launch an HTML page linking to several well-
written R manuals such as: ”An Introduction to R”, ”The R Language Deﬁ-
nition”, ”R Installation and Administration”, and ”R Data Import/Export”.
Further help can be obtained from http://cran.r-project.org. Its ”con-
tributed” page contains well-written freely available on-line books
3
and use-
ful reference charts
4
. At http://www.r-project.org you can use R site
2
Import/Export manual”.
3
”R for Beginners” by Emmanuel Paradis or the ”The R Guide” by Jason Owen
4
”R reference card” by Tom Short or by Jonathan Baron
4 CHAPTER 1. BRIEF INTRODUCTION INTO USING R
search, Rseek, or other useful search engines. There are a number of useful
URL’s with information on R.
5
1.3 Calculating with R
R can be used as a simple calculator. For instance, to add 2 and 3 we simply
insert the following.
> 2+3
[1] 5
In many calculations the natural base e = 2.718282 of exponential functions
is used. Such type of functions can be called as follows.
> exp(1)
[1] 2.718282
To compute e
2
= e · e we use exp(2).
6
So, indeed, we have e
x
=exp(x), for
any value of x.
The sum 1 + 2 + 3 + 4 + 5 can be computed by
> sum(1:5)
[1] 15
and the product 5! = 5 · 4 · 3 · 2 · 1 by
> prod(1:5)
[1] 120
1.4 Generating a sequence and a factor
In order to compute so-called quantiles of distributions (see e.g. Section
2.1.4) or plots of functions, we need to generate sequences of numbers. The
easiest way to construct a sequence of numbers is by
> 1:5
[1] 1 2 3 4 5
5
We mention in particular:
http://faculty.ucr.edu/
~
tgirke/Documents/R_BioCond/R_BioCondManual.html
6
The argument of functions is always placed between parenthesis ().
1.5. COMPUTING ON A DATA VECTOR 5
This sequence can also be produced by the function seq, which allows for
various sizes of steps to be chosen. For instance, in order to compute per-
centiles of a distribution we may want to generate numbers between zero and
one with step size equal to 0.1.
> seq(0,1,0.1)
[1] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
For plotting and testing of hypotheses we need to generate yet another
type of sequence, called a “factor”. It is designed to indicate an experimen-
tal condition of a measurement or the group to which a patient belongs.
7
When, for instance, for each of three experimental conditions there are mea-
surements from ﬁve patients, the corresponding factor can be generated as
follows.
> factor <- gl(3,5)
> factor
[1] 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3
Levels: 1 2 3
The three conditions are often called “levels” of a factor. Each of these
levels has ﬁve repeats corresponding to the number of observations (patients)
within each level (type of disease). We shall further illustrate the idea of a
factor soon because it is very useful for purposes of visualization.
1.5 Computing on a data vector
A data vector is simply a collection of numbers obtained as outcomes from
measurements. This can be illustrated by a simple example on expression
values of a gene. Suppose that gene expression values 1, 1.5, and 1.25 from
the persons ”Eric”, ”Peter”, and ”Anna” are available. To store these in a
vector we use the concatenate command c(), as follows.
> gene1 <- c(1.00,1.50,1.25)
> gene1
[1] 1.00 1.50 1.25
7
See e.g. Samuals & Witmer (2003, Chap. 8) for a full explanation of experiments
and statistical principles of design.
6 CHAPTER 1. BRIEF INTRODUCTION INTO USING R
Now we have created the object gene1 containing three gene expression val-
ues. To compute the sum, mean, and standard deviation of the gene expres-
sion values we use the corresponding built-in-functions.
> sum(gene1)
[1] 3.75
> mean(gene1)
[1] 1.25
> sum(gene1)/3
[1] 1.25
> sd(gene1)
[1] 0.25
> sqrt(sum((gene1-mean(gene1))^2)/2)
[1] 0.25
By deﬁning x
1
= 1.00, x
2
= 1.50, and x
3
= 1.25, the sum of the weights can
be expressed as

n
i=1
x
i
= 3.75. The mathematical summation symbol

is
in R language simply sum. The mean is denoted by x =

3
i=1
x
i
/3 = 1.25
and the sample standard deviation as
s =
¸
¸
¸
_
3

i=1
(x
i
−x)
2
/(3 −1) = 0.25.
1.6 Constructing a data matrix
In various types of spreadsheets it is custom to store data values in the
form of a matrix consisting of rows and columns. In bioinformatics gene
expression values (from several groups of patients) are stored as rows such
that each row contains the expressions values of the patients corresponding
to a particular gene and each column contains all gene expression values for
a particular person. To illustrate this by a small example suppose that we
have the following expression values on three genes from Eric, Peter, and
Anna.
8
> gene2 <- c(1.35,1.55,1.00)
> gene3 <- c(-1.10,-1.50,-1.25)
> gene4 <- c(-1.20,-1.30,-1.00)
8
By the function data.entry you can open and edit a screen with the values of a
matrix.
1.6. CONSTRUCTING A DATA MATRIX 7
Before constructing the matrix it is convenient to add the names of the rows
and the columns. To do so we construct the following list.
> rowcolnames <- list(c("gene1","gene2","gene3","gene4"),
+ c("Eric","Peter","Anna"))
After the last comma in the ﬁrst line we give a carriage return for R to come
up with a new line starting with + in order to complete a command. Now we
can construct a matrix containing the expression values from our four genes,
as follows.
> gendat <- matrix(c(gene1,gene2,gene3,gene4), nrow=4, ncol=3,
+ byrow=TRUE, dimnames = rowcolnames)
Here, nrow indicates the number of rows and ncol the number of columns.
The gene vectors are placed in the matrix as rows. The names of the rows
and columns are attached by the dimnames parameter. To see the content of
the just created object gendat, we print it to the screen.
> gendat
Eric Peter Anna
gene1 1.00 1.50 1.25
gene2 1.35 1.55 1.30
gene3 -1.10 -1.50 -1.25
gene4 -1.20 -1.30 -1.00
A matrix such as gendat has two indices [i,j], the ﬁrst of which refers to
rows and the second to columns
9
. Thus, if you want to print the second
element of the ﬁrst row to the screen, then type gendat[1,2]. If you want
to print the ﬁrst row, then use gendat[1,]. For the second column, use
gendat[,2].
It may be desirable to write the data to a ﬁle for using these in a later
stage or to send these to a college of yours. Consider the following script.
> write.table(gendat,file="D:/data/gendat.Rdata")
Eric Peter Anna
gene1 1.00 1.50 1.25
9
Indices referring to rows, columns, or elements are always between square brackets [].
8 CHAPTER 1. BRIEF INTRODUCTION INTO USING R
gene2 1.35 1.55 1.30
gene3 -1.10 -1.50 -1.25
gene4 -1.20 -1.30 -1.00
An alternative is to use write.csv.
10
1.7 Computing on a data matrix
Means or standard deviations of rows or columns are often important for
drawing biologically relevant conclusions. Such type of computations on a
data matrix can be accomplished by “for loops”. However, it is much more
convenient to use the apply functionality on a matrix. To do so we specify
the name of the matrix, indicate rows or columns (1 for rows and 2 for
columns), and the name of the function. To illustrate this we compute the
mean of each person (column).
> apply(gendat,2,mean)
Eric Peter Anna
0.0125 0.0625 0.0750
Similarly, the mean of each gene (row) can be computed.
> apply(gendat,1,mean)
gene1 gene2 gene3 gene4
1.250000 1.400000 -1.283333 -1.166667
It frequently happens that we want to re-order the rows of a matrix according
to a certain criterion, or, more speciﬁcally, the values in a certain column
vector. For instance, to re-order the matrix gendat according to the row
means, it is convenient to store these in a vector and to use the function
order.
> meanexprsval <- apply(gendat,1,mean)
> o <- order(meanexprsval,decreasing=TRUE)
> o
[1] 2 1 4 3
10
For more see the ”R Data import/Export” manual, Chapter 3 of the book ”R for
Beginners”, or search the internet by the key ”r wiki matrix”.
1.7. COMPUTING ON A DATA MATRIX 9
Thus gene2 appears ﬁrst because it has the largest mean 1.4, then gene1
with 1.25, followed by gene4 with -1.16 and, ﬁnally, gene3 with -1.28. Now
that we have collected the order numbers in the vector o, we can re-order
the whole matrix by specifying o as the row index.
11
> gendat[o,]
Eric Peter Anna
gene2 1.35 1.55 1.30
gene1 1.00 1.50 1.25
gene4 -1.20 -1.30 -1.00
gene3 -1.10 -1.50 -1.25
Another frequently occurring problem is that of selecting genes with a certain
property. We illustrate this by several methods to select genes with positive
mean expression values. A ﬁrst method starts with the observation that the
ﬁrst two rows have positive means and to use c(1,2) as a row index.
> gendat[c(1,2),]
Eric Peter Anna
gene1 1.00 1.50 1.25
gene2 1.35 1.55 1.30
A second way is to use the row names as an index.
> gendat[c("gene1","gene2"),]
Eric Peter Anna
gene1 1.00 1.50 1.25
gene2 1.35 1.55 1.30
A third and more advanced way is to use an evaluation in terms of TRUE
or FALSE of logical elements of a vector. For instance, we may evaluate
whether the row mean is positive.
> meanexprsval > 0
gene1 gene2 gene3 gene4
TRUE TRUE FALSE FALSE
Now we can use the evaluation of meanexprsval > 0 in terms of the values
TRUE or FALSE as a row index.
11
You can also use functions like sort or rank.
10 CHAPTER 1. BRIEF INTRODUCTION INTO USING R
> gendat[meanexprsval > 0,]
Eric Peter Anna
gene1 1.00 1.50 1.25
gene2 1.35 1.55 1.30
Observe that this selects genes for which the evaluation equals TRUE. This
illustrates that genes can be selected by their row index, row name or value
on a logical variable.
1.8 Application to the Golub (1999) data
The gene expression data collected by Golub et al. (1999) are among the clas-
sical in bioinformatics. A selection of the set is called golub and is contained
in the multtest package, which is part of Bioconductor. The data consist
of gene expression values of 3051 genes (rows) from 38 leukemia patients
12
.
Twenty seven patients are diagnosed as acute lymphoblastic leukemia (ALL)
and eleven as acute myeloid leukemia (AML). The tumor class is given by
the numeric vector golub.cl, where ALL is indicated by 0 and AML by
1. The gene names are collected in the matrix golub.gnames of which the
columns correspond to the gene index, ID, and Name, respectively. We shall
ﬁrst concentrate on expression values of a gene with manufacturer name
"M92287_at", which is known in biology as "CCND3 Cyclin D3". The ex-
pression values of this gene are collected in row 1042 of golub. To load the
data and to obtain relevant information from row 1042 of golub.gnames, use
the following.
> library(multtest); data(golub)
> golub.gnames[1042,]
[1] "2354" "CCND3 Cyclin D3" "M92287_at"
The data are stored in a matrix called golub. The number of rows and
columns can be obtained by the functions nrow and ncol, respectively.
> nrow(golub)
[1] 3051
> ncol(golub)
[1] 38
12
The data are pre-processed by procedures described in Dudoit et al. (2002).
1.8. APPLICATION TO THE GOLUB (1999) DATA 11
So the matrix has 3051 rows and 38 columns, see also dim(golub). Each
data element has a row and a column index. Recall that the ﬁrst index refers
to rows and the second to columns. Hence, the second value from row 1042
can be printed to the screen as follows.
> golub[1042,2]
[1] 1.52405
So 1.52405 is the expression value of gene CCND3 Cyclin D3 from patient
number 2. The values of the ﬁrst column can be printed to the screen by the
following.
> golub[,1]
To save space the output is not shown. We may now print the expression
values of gene CCND3 Cyclin D3 (row 1042) to the screen.
> golub[1042,]
[1] 2.10892 1.52405 1.96403 2.33597 1.85111 1.99391 2.06597 1.81649
[9] 2.17622 1.80861 2.44562 1.90496 2.76610 1.32551 2.59385 1.92776
[17] 1.10546 1.27645 1.83051 1.78352 0.45827 2.18119 2.31428 1.99927
[25] 1.36844 2.37351 1.83485 0.88941 1.45014 0.42904 0.82667 0.63637
[33] 1.02250 0.12758 -0.74333 0.73784 0.49470 1.12058
To print the expression values of gene CCND3 Cyclin D3 to the screen only
for the ALL patients, we have to refer to the ﬁrst twenty seven elements of
row 1042. A possibility to do so is by the following.
> golub[1042,1:27]
However, for the work ahead it is much more convenient to construct a factor
indicating the tumor class of the patients. This will turn out useful e.g.
for separating the tumor groups in various visualization procedures. The
factor will be called gol.fac and is constructed from the vector golub.cl,
as follows.
> gol.fac <- factor(golub.cl, levels=0:1, labels = c("ALL","AML"))
In the sequel this factor will be used frequently. Obviously, the labels corre-
spond to the two tumor classes. The evaluation of gol.fac=="ALL" returns
TRUE for the ﬁrst twenty seven values and FALSE for the remaining eleven.
This is useful as a column index for selecting the expression values of the
ALL patients. The expression values of gene CCND3 Cyclin D3 from the
ALL patients can now be printed to the screen, as follows.
12 CHAPTER 1. BRIEF INTRODUCTION INTO USING R
> golub[1042,gol.fac=="ALL"]
For many types of computations it is very useful to combine a factor with
the apply functionality. For instance, to compute the mean gene expression
over the ALL patients for each of the genes, we may use the following.
> meanALL <- apply(golub[,gol.fac=="ALL"], 1, mean)
The speciﬁcation golub[,gol.fac=="ALL"] selects the matrix with gene ex-
pressions corresponding to the ALL patients. The 3051 means are assigned
to the vector meanALL.
After reading the classical article by Golub et al. (1999), which is strongly
recommended, one becomes easily interested in the properties of certain
genes. For instance, gene CD33 plays an important role in distinguishing
lymphoid from myeloid lineage cells. To perform computations on the ex-
pressions of this gene we need to know its row index. This can obtained by
the grep function.
13
> grep("CD33",golub.gnames[,2])
[1] 808
Hence, the expression values of antigen CD33 are available at golub[808,]
and further information on it by golub.gnames[808,].
1.9 Running scripts
It is very convenient to use a plain text writer like Notepad, Kate, Emacs, or
WinEdt for the formulation of several consecutive R commands as separated
lines (scripts). Such command lines can be executed by simply using copy
and paste into the command line editor of R. Another possibility is to execute
a script from a ﬁle. To illustrate the latter consider the following.
> library(multtest); data(golub)
> gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
> mall <- apply(golub[,gol.fac=="ALL"], 1, mean)
> maml <- apply(golub[,gol.fac=="AML"], 1, mean)
> o <- order(abs(mall-maml), decreasing=TRUE)
> print(golub.gnames[o[1:5],2])
13
Indeed, several functions of R are inspired by the Linux operating system.
1.10. OVERVIEW AND CONCLUDING REMARKS 13
[1] "CST3 Cystatin C (amyloid angiopathy and cerebral hemorrhage)"
[2] "INTERLEUKIN-8 PRECURSOR"
[3] "Interleukin 8 (IL8) gene"
[4] "DF D component of complement (adipsin)"
[5] "MPO Myeloperoxidase"
The row means of the expression values per patient group are computed and
stored in the object mall and maml, respectively. The absolute values of the
diﬀerences in means are computed and their order numbers (from large to
small) are stored in the vector o. Next, the names of the ﬁve genes with the
largest diﬀerences in mean are printed to the screen.
After saving the script under e.g. the name meandif.R in the directory
D:\\Rscripts\\meandif.R, it can be executed by using source("D:\\Rscripts\\meandif.R").
Once the script is available for a typewriter it is easy to adapt it and to re-run
it.
Readers are strongly recommended to trial-and-error with respect to writ-
ing programming scripts. To run these it is very convenient to have your
favorite word processor available and to use, for instance, the copy-and-paste
functionality.
1.10 Overview and concluding remarks
It is easy to install R and Bioconductor. R has many convenient built-in-
functions for statistical programming. Help and illustrations on many topics
are available from various sources. With the reference charts, R manuals,
(on-line) books and R Wiki at hand you have various sources of information
several GUI’s available, we shall concentrate on the command line editor
because its range of possibilities is much larger.
The above introduction is of course very brief. A more extensive in-
troduction into R, assuming some background on biomedical statistics, is
given by Dalgaard (2002). There are book length treatments combining R
with statistics (Venables, & Ripley, 2002; Everitt & Hothorn, 2006). Other
treatments go much deeper into programming aspects (Becker, Chambers, &
Wilks, 1988; Venables & Ripley, 2000; Gentleman, 2008).
For the sake of illustration we shall work frequently with the data kindly
provided by Golub et al. (1999) and Chiaretti et al. (2004). The corre-
14 CHAPTER 1. BRIEF INTRODUCTION INTO USING R
sponding scientiﬁc articles are freely available from the web. Having these
1.11 Exercises
1. Some questions to orientate yourself.
(a) Use the function class to ﬁnd the class to which the follow-
ing objects belong: golub, golub[1,1],golub.cl, golub.gnames,
apply, exp, gol.fac, plot, ALL.
(b) What is the meaning of the following abbreviations: rm, sum, prod,
seq, sd, nrow.
(c) For what purpose are the following functions useful: grep, apply,
gl, library, source, setwd, history, str.
2. gendat Consider the data in the matrix gendat, constructed in Sec-
tion 1.6. Its small size has the advantage that you can check your
computations even by a pocket calculator.
14
(a) Use apply to compute the standard deviation of the persons.
(b) Use apply to compute the standard deviation of the genes.
(c) Order the matrix according to the gene standard deviations.
(d) Which gene has the largest standard deviation?
3. Computations on gene means of the Golub data.
(a) Use apply to compute the mean gene expression value.
(b) Order the data matrix according to the gene means.
(c) Give the names of the three genes with the largest mean expression
value.
(d) Give the biological names of these genes.
4. Computations on gene standard deviations of the Golub data.
(a) Use apply to compute the standard deviation per gene.
14
Obtaining some routine with the apply functionality is quite helpful for what follows.
1.11. EXERCISES 15
(b) Select the expression values of the genes with standard deviation
larger than two.
(c) How many genes have this property?
5. Oncogenes in Golub data.
(a) How many oncogenes are there in the dataset? Hint: Use grep.
(b) Find the biological names of the three oncogenes with the largest
mean expression value for the ALL patients.
(c) Do the same for the AML patients.
(d) Write the gene probe ID and the gene names of the ten genes with
largest mean gene expression value to a csv ﬁle.
6. Constructing a factor. Construct factors that correspond to the follow-
ing setting.
(a) An experiment with two conditions each with four measurements.
(b) Five conditions each with three measurements.
(c) Three conditions each with ﬁve measurements.
7. Gene means for B1 patients. Load the ALL data from the ALL library
and use str and openVignette() for a further orientation.
(a) Use exprs(ALL[,ALL$BT=="B1"] to extract the gene expressions from the patients in disease stage B1. Compute the mean gene expressions over these patients. (b) Give the gene identiﬁers of the three genes with the largest mean. 16 CHAPTER 1. BRIEF INTRODUCTION INTO USING R Chapter 2 Data Display and Descriptive Statistics A few essential methods are given to display and visualize data. It quickly answers questions like: How are my data distributed? How can the frequen- cies of nucleotides from a gene be visualized? Are there outliers in my data? Does the distribution of my data resemble that of a bell-shaped curve? Are there diﬀerences between gene expression values taken from two groups of patients? The most important central tendencies (mean, median) are deﬁned and illustrated together with the most important measures of spread (standard deviation, variance, inter quartile range, and median absolute deviation). 2.1 Univariate data display To observe the distribution of data various visualization methods are made available. These are frequently used by practitioners as well as by experts. 2.1.1 Frequency table Discrete data occur when the values naturally fall into categories. A fre- quency table simply gives the number of occurrences within a category. Example 1. A gene consists of a sequence of nucleotides {A, C, G, T}. The number of each nucleotide can be displayed in a frequency table. This 17 18 CHAPTER 2. DATA DISPLAY AND DESCRIPTIVE STATISTICS will be illustrated by the Zyxin gene which plays an important role in cell adhesion (Golub et al., 1999). The accession number (X94991.1) of one of its variants can be found in a data base like NCBI (UniGene). The code below illustrates how to read the sequence ”X94991.1” of the species homo sapiens from GenBank, , to construct a pie from a frequency table of the four nucleotides. install.packages(c("ape"),repo="http://cran.r-project.org",dep=TRUE) library(ape) table(read.GenBank(c("X94991.1"),as.character=TRUE)) pie(table(read.GenBank(c("X94991.1")))) From the resulting frequencies in Table 2.1 it seems that the nucleotides are not equally likely. A nice way to visualize a frequency table is by plotting a pie. Table 2.1: A frequency table and its pie of Zyxin gene. A C G T 410 789 573 394 a c g t 2.1.2 Plotting data An elementary method to visualize data is by using a so-called stripchart, by which the values of the data are represented as e.g. small boxes. Often, 2.1. UNIVARIATE DATA DISPLAY 19 it is useful in combination with a factor that distinguishes members from diﬀerent experimental conditions or patients groups. Example 1. Many visualization methods will be illustrated by the Golub et al. (1999) data. We shall concentrate on the expression values of gene "CCND3 Cyclin D3", which are collected in row 1042 of the data matrix golub. To plot the data values one can simply use plot(golub[1042,]). In the resulting plot in Figure 2.1 the vertical axis gives the size of the expression values and the horizontal axis the index of the patients. It can be observed that the values for patient 28 to 38 are somewhat lower, but, indeed, the picture is not very clear because the groups are not plotted separately. To produce two adjacent stripcharts one for the ALL and one for the AML patients, we use the factor called gol.fac from the previous chapter. data(golub, package = "multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) stripchart(golub[1042,] ~ gol.fac, method="jitter") From the resulting Figure 2.2 it can be observed that the CCND3 Cyclin D3 expression values of the ALL patients tend to have larger expression values than those of the AML patients. 2.1.3 Histogram Another method to visualize data is by dividing the range of data values into a number of intervals and to plot the frequency per interval as a bar. Such a plot is called a histogram. Example 1. A histogram of the expression values of gene "CCND3 Cyclin D3" of the acute lymphoblastic leukemia patients can be produced as follows. > hist(golub[1042, gol.fac=="ALL"]) The function hist divides the data into 5 intervals having width equal to 0.5, see Figure 2.3. Observe from the latter that one value is small and the other are more or less symmetrically distributed around the mean. 20 CHAPTER 2. DATA DISPLAY AND DESCRIPTIVE STATISTICS 0 10 20 30 0 . 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 Index g o l u b [ 1 0 4 2 , ] Figure 2.1: Plot of gene ex- pression values of CCND3 Cyclin D3. ALL AML 0 . 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 Figure 2.2: Stripchart of gene expression values of CCND3 Cyclin D3 for ALL and AML patients. 2.1.4 Boxplot It is always possible to sort n data values to have increasing order x 1 ≤ x 2 · · · ≤ x n , where x 1 is the smallest, x 2 is the ﬁrst-to-the smallest, etc. Let x 0.25 be a number for which it holds that 25% of the data values x 1 , · · · , x n is smaller. That is, 25% of the data values lay on the left side of the number x 0.25 , reason for which it is called the ﬁrst quartile or the 25th percentile. The second quartile is the value x 0.50 such that 50% of the data values are smaller. Similarly, the third quartile or 75th percentile is the value x 0.75 such that 75% of the data is smaller. A popular method to display data is by drawing a box around the ﬁrst and the third quartile (a bold line segment for the median), and the smaller line segments (whiskers) for the smallest and the largest data values. Such a data display is known as a box-and-whisker plot. Example 1. A vector with gene expression values can be put into in- creasing order by the function sort. We shall illustrate this by the ALL 2.1. UNIVARIATE DATA DISPLAY 21 expression values of gene "CCND3 Cyclin D3" in row 1042 of golub. > x <- sort(golub[1042, gol.fac=="ALL"], decreasing = FALSE) > x[1:5] [1] 0.458 1.105 1.276 1.326 1.368 The second command prints the ﬁrst ﬁve values of the sorted data values to the screen, so that we have x 1 = 0.458, x 2 = 1.105, etc. Note that the mathematical notation x i corresponds exactly to the R notation x[i] Histogram of golub[1042, gol.fac == "ALL"] golub[1042, gol.fac == "ALL"] F r e q u e n c y 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 2 4 6 8 1 0 1 2 Figure 2.3: Histogram of ALL ex- pression values of gene CCND3 Cyclin D3. ALL AML 0 . 5 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 Figure 2.4: Boxplot of ALL and AML expression values of gene CCND3 Cyclin D3. Example 2. A view on the distribution of the expression values of the ALL and the AML patients on gene CCND3 Cyclin D3 can be obtained by constructing two separate boxplots adjacent to one another. To produce such a plot the factor gol.fac is again very useful. > boxplot(golub[1042,] ~ gol.fac) From the position of the boxes in Figure 2.4 it can be observed that the gene expression values for ALL are larger than those for AML. Furthermore, since the two sub-boxes around the median are more or less equally wide, the data are quite symmetrically distributed around the median. 22 CHAPTER 2. DATA DISPLAY AND DESCRIPTIVE STATISTICS To compute exact values for the quartiles we need a sequence running from 0.00 to 1.00 with steps equal to 0.25. To construct such a sequence the function seq is useful. > pvec <- seq(0,1,0.25) > quantile(golub[1042, gol.fac=="ALL"],pvec) 0% 25% 50% 75% 100% 0.458 1.796 1.928 2.179 2.766 The ﬁrst quartile x 0.25 = 1.796, the second x 0.50 = 1.928, and the third x 0.75 = 2.179. The smallest observed expression value equals x 0.00 = 0.458 and the largest x 1.00 = 2.77. The latter can also be obtained by the function min(golub[1042, gol.fac=="ALL"]) and max(golub[1042, gol.fac=="ALL"]), or more brieﬂy by range(golub[1042, gol.fac=="ALL"]). Outliers are data values laying far apart from the pattern set by the majority of the data values. The implementation in R of the (modiﬁed) boxplot draws such outlier points separately as small circles. A data point x is deﬁned as an outlier point if x < x 0.25 −1.5 · (x 0.75 −x 0.25 ) or x > x 0.75 + 1.5 · (x 0.75 −x 0.25 ). From Figure 2.4 it can be observed that there are outliers among the gene expression values of ALL patients. These are the smaller values 0.45827 and 1.10546, and the largest value 2.76610. The AML expression values have one outlier with value -0.74333. To deﬁne extreme outliers, the factor 1.5 is raised to 3.0. Note that this is a descriptive way of deﬁning outliers instead of statistically testing for the existence of an outlier. 2.1.5 Quantile-Quantile plot A method to visualize the distribution of gene expression values is by the so-called quantile-quantile (Q-Q) plot. In such a plot the quantiles of the gene expression values are displayed against the corresponding quantiles of the normal (bell-shaped). A straight line is added representing points which correspond exactly to the quantiles of the normal distribution. By observing the extent in which the points appear on the line, it can be evaluated to what degree the data are normally distributed. That is, the closer the gene 2.1. UNIVARIATE DATA DISPLAY 23 expression values appear to the line, the more likely it is that the data are normally distributed. −2 −1 0 1 2 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 Normal Q−Q Plot Theoretical Quantiles S a m p l e Q u a n t i l e s Figure 2.5: Q-Q plot of ALL gene expression values of CCND3 Cyclin D3. Example 1. To produce a Q-Q plot of the ALL gene expression values of CCND3 Cyclin D3 one may use the following. qqnorm(golub[1042, gol.fac=="ALL"]) qqline(golub[1042, gol.fac=="ALL"]) From the resulting Figure 2.5 it can be observed that most of the data points are on or near the straight line, while a few others are further away. The above example illustrates a case where the degree of non-normality is moderate so that a clear conclusion cannot be drawn. By making the 24 CHAPTER 2. DATA DISPLAY AND DESCRIPTIVE STATISTICS exercises below, the reader will gather more experience with the degree in which gene expression values are normally distributed. 2.2 Descriptive statistics There exist various ways to describe the central tendency as well as the spread of data. In particular, the central tendency can be described by the mean or the median, and the spread by the variance, standard deviation, interquartile range, or median absolute deviation. These will be deﬁned and illustrated. 2.2.1 Measures of central tendency The most important descriptive statistics for central tendency are the mean and the median. The sample mean of the data values x 1 , · · · , x n is deﬁned as x = 1 n n i=1 x i = 1 n (x 1 +· · · + x n ). Thus the sample mean is simply the average of the n data values. Since it is the sum of all data values divided by the sample size, a few extreme data values may largely inﬂuence its size. In other words, the mean is not robust against outliers. The median is deﬁned as the second quartile or the 50th percentile, and is denoted by x 0.50 . When the data are symmetrically distributed around the mean, then the mean and the median are equal. Since extreme data values do not inﬂuence the size of the median, it is very robust against outliers. Robustness is important in bioinformatics because data are frequently con- taminated by extreme or otherwise inﬂuential data values. Example 1. To compute the mean and median of the ALL expression values of gene CCND3 Cyclin D3 consider the following. > mean(golub[1042, gol.fac=="ALL"]) [1] 1.89 > median(golub[1042, gol.fac=="ALL"]) [1] 1.93 2.2. DESCRIPTIVE STATISTICS 25 Note that the mean and the median do not diﬀer much so that the distribu- tion seems quite symmetric. 2.2.2 Measures of spread The most important measures of spread are the standard deviation, the in- terquartile range, and the median absolute deviation. The standard deviation is the square root of the sample variance, which is deﬁned as s 2 = 1 n −1 n i=1 (x i −x) 2 = 1 n −1 _ (x 1 −x) 2 +· · · + (x n −x) 2 _ . Hence, it is the average of the squared diﬀerences between the data values and the sample mean. The sample standard deviation s is the square root of the sample variance and may be interpreted as the distance of the data values to the mean. The variance and the standard deviation are not robust against outliers. The interquartile range is deﬁned as the diﬀerence between the third and the ﬁrst quartile, that is x 0.75 − x 0.25 . It can be computed by the function IQR(x). More speciﬁcally, the value IQR(x)/1.349 is a robust estimator of the standard deviation. The median absolute deviation (MAD) is deﬁned as a constant times the median of the absolute deviations of the data from the median (e.g. Jureˇckov´ a & Picek, 2006, p. 63). In R it is computed by the function mad deﬁned as the median of the sequence |x 1 −x 0.50 |, · · · , |x n −x 0.50 | multiplied by the constant 1.4826. It equals the standard deviation in case the data come from a bell-shaped (normal) distribution (see Section 3.2.1). Because the interquartile range and the median absolute deviation are based on quantiles, these are robust against outliers. Example 1. These measures of spread for the ALL expression values of gene CCND3 Cyclin D3 can be computed as follows. > sd(golub[1042, gol.fac=="ALL"]) [1] 0.491 > IQR(golub[1042, gol.fac=="ALL"]) / 1.349 [1] 0.284 > mad(golub[1042, gol.fac=="ALL"]) [1] 0.368 26 CHAPTER 2. DATA DISPLAY AND DESCRIPTIVE STATISTICS Due to the three outliers (cf. Figure 2.4) the standard deviation is larger than the interquartile range and the mean absolute deviation. That is, the absolute diﬀerences with respect to the median are somewhat smaller, than the root of the squared diﬀerences. 2.3 Overview and concluding remarks Data can be stored as a vector or a data matrix on which various useful functions are deﬁned. In particular, it is easy to produce a pie, histogram, boxplot, or Q-Q plot of a vector of data. These plots give a useful ﬁrst impression of the degree of (non)normality of gene expression values. To construct the histogram used the default method to compute the num- ber of bars or breaks. If the data are distributed according to a bell-shaped curve, then this is often a good strategy. The number of bars can be chosen by the breaks option of the function hist. Optimal choices for this are dis- cussed by e.g. Venables and Ripley (2002). 2.4 Exercises Since the majority of the exercises are based on the Golub et al. (1999) data, it is essential to make these available and to learn to work with it. To stimulate self-study the answers are given at the end of the book. 1. Illustration of mean and standard deviation. (a) Compute the mean and the standard deviation for 1, 1.5, 2, 2.5, 3. (b) Compute the mean and the standard deviation for 1, 1.5, 2, 2.5, 30. (c) Comment on the diﬀerences. 2. Comparing normality for two genes. Consider the gene expression val- ues in row 790 and 66 of the Golub et al. (1999) data. (a) Produce a boxplot for the expression values of the ALL patients and comment on the diﬀerences. Are there outliers? 2.4. EXERCISES 27 (b) Produce a QQ-plot and formulate a hypothesis about the normal- ity of the genes. (c) Compute the mean and the median for the expression values of the ALL patients and compare these. Do this for both genes. 3. Eﬀect size. An important statistic to measure the eﬀect size which is deﬁned for a sample as x/s. It measures the mean relative to the standard deviation, so that is value is large when the mean is large and the standard deviation small. (a) Determine the ﬁve genes with the largest eﬀect size of the ALL patients from the Golub et al. (1999) data. Comment on their size. (b) Invent a robust variant of the eﬀect size and use it to answer the previous question. 4. Plotting gene expressions "CCND3 Cyclin D3". Use the gene expres- sions from "CCND3 Cyclin D3" of Golub et al. (1999) collected in row 1042 of the object golub from the multtest library. After using the function plot you produce an object on which you can program. (a) Produce a so-called stripchart for the gene expressions separately for the ALL as well as for the AML patients. Hint: Use a factor for appropriate separation. (b) Rotate the plot to a vertical position and keep it that way for the questions to come. (c) Color the ALL expressions red and AML blue. Hint: Use the col parameter. (d) Add a title to the plot. Hint: Use title. (e) Change the boxes into stars. Hint: Use the pch parameter. Hint: Store the ﬁnal script you like the most in your typewriter in order to be able to use it eﬃciently later on. 5. Box-and-Whiskers plot of "CCND3 Cyclin D3". Use the gene expres- sions "CCND3 Cyclin D3" of Golub et al. (1999) from row 1042 of the object golub of the multtest library. (a) Construct the boxplot in Figure 2.6. 28 CHAPTER 2. DATA DISPLAY AND DESCRIPTIVE STATISTICS (b) Add text to the plot to explain the meaning of the upper and lower part of the box. (c) Do the same for the wiskers. (d) Export your plot to eps format. Hint 1: Use locator() to ﬁnd coordinates of the position of the plot. Hint 2: Use xlim to make the plot somewhat wider. Hint 3: Use arrows to add an arrow. Hint 4: Use text to add information at a certain position. 6. Box-and-wiskers plot of persons of Golub et al. (1999) data. (a) Use boxplot(data.frame(golub)) to produce a box-and-wiskers plot for each column (person). Make a screen shot to save it in a word processor. Describe what you see. Are the medians of similar size? Is the inter quartile range more or less equal. Are there outliers? (b) Compute the mean and medians of the persons. What do you observe? (c) Compute the range (minimal and maximum value) of the standard deviations, the IQR and MAD of the persons. Comment of what you observe. 7. Oncogenes of Golub et al. (1999) data. (a) Select the oncogens by the grep facility and produce a box-and- wiskers plot of the gene expressions of the ALL patients. (b) Do the same for the AML patients and use par(mfrow=c(2,1)) to combine the two plots such that the second is beneath the ﬁrst. Are there genes with clear diﬀerences between the groups? 8. Descriptive statistics for the ALL gene expression values of the Golub et al. (1999) data. (a) Compute the mean and median for gene expression values of the ALL patients, report their range and comment on it. (b) Compute the SD, IQR, and MAD for gene expression values of the ALL patients, report their range and comment on it. 2.4. EXERCISES 29 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 Median Outlier Figure 2.6: Boxplot with arrows and explaining text. 30 CHAPTER 2. DATA DISPLAY AND DESCRIPTIVE STATISTICS Chapter 3 Important Distributions Questions that concern us in this chapter are: What is the probability to ﬁnd fourteen purines in a microRNA of length twenty two? If expressions from ALL patients of gene CCND3 Cyclin D3 are normally distributed with mean 1.90 and standard deviation 0.5, what is the probability to observe expression values larger than 2.4? To answer such type of questions we need to know more about statis- tical distributions (e.g. Samuels & Witmer, 2003). In this chapter several important distributions will be deﬁned, explained, and illustrated. In par- ticular, the discrete distribution binomial and the continuous distributions normal, T, F, and chi-squared will be elaborated. These distributions have a wealth of applications to statistically testing biological hypotheses. Only when deemed relevant, the density function, the distribution function, the mean µ (mu), and the standard deviation σ (sigma), are explicitly deﬁned. 3.1 Discrete distributions The binomial distribution is fundamental and has many applications in medicine and bioinformatics. 3.1.1 Binomial distribution The binomial distribution ﬁts to repeated trials each with a dichotomous out- come such as succes-failure, healthy-disease, heads-tails, purine-pyrimidine, etc. When there are n trials, then the number of ways to obtain k successes 31 32 CHAPTER 3. IMPORTANT DISTRIBUTIONS out of n is given by the binomial coeﬃcient n! k!(n −k)! , where n! = n · (n − 1) · · · 1 and 0! = 1 (Samuels & Witmer, 2003). The binomial probability of k successes out of n consists of the product of this coeﬃcient with the probability of k successes and the probability of n − k failures. Let p be the probability of succes in a single trial and X the (random) variable denoting the number of successes. Then the probability P of the event (X = k) that k successes occur out of n trails can be expressed as P(X = k) = n! k!(n −k)! p k (1 −p) n−k , for k = 0, · · · , n. (3.1) The collection of these probabilities is called the probability density function. 1 Example 1. To visualize the Binomial distribution, load the TeachingDemos package and use the command vis.binom(). Click on ”Show Normal Ap- proximation” and observe that the approximation improves as n increases, taking p for instance near 0.5. Example 2. If two carriers of the gen for albinism marry, then each of the children has probability of 1/4 of being albino. What is the probability for one child out of three to be albino? To answer this question we take n = 3, k = 1, and p = 0.25 into Equation (3.1) and obtain P(X = 1) = 3! 1!(3 −1)! 0.25 1 0.75 2 = 3 · 0.140625 = 0.421875. An elementary manner to compute this in R is by > choose(3,1)* 0.25^1* 0.75^2 where choose(3,1) computes the binomial coeﬃcient. It is more eﬃcient to compute this by the built-in-density-function dbinom(k,n,p), for instance to print the values of the probabilities. 1 For a binomially distributed variable np is the mean, np(1 − p) the variance, and _ np(1 −p) the standard deviation. 3.1. DISCRETE DISTRIBUTIONS 33 > for (k in 0:3) print(dbinom(k,3,0.25)) Changing d into p yields the so-called distribution function with the cumula- tive probabilities. That is, the probability that the number of Heads is lower than or equal to two P(X ≤ 2) is computed by pbinom(2,3,0.25). The values of the density and distribution function are summarized in Table 3.1. From the table we read that the probability of no albino child is 0.4218 and the probability that all three children are albino equals 0.0156. Table 3.1: Discrete density and distribution function values of S 3 , with p = 0.6. number of Heads k = 0 k = 1 k = 2 k = 3 density P(X = k) 0.4218 0.4218 0.1406 0.0156 distribution P(X ≤ k) 0.4218 0.843 0.9843 1 Example 3. RNA consists of a sequence of nucleotides A, G, U, and C, where the ﬁrst two are purines and the last two are pyrimidines. Suppose, for the purpose of illustration, that the length of a certain micro RNA is 22, that the probability of a purine equals 0.7, and that the process of placing purines and pyrimidines is binomially distributed. The event that our microRNA contains 14 purines can be represented by X = 14. The probability of this event can be computed by P(X = 14) = 22! 14!(22 −14)! 0.7 14 0.3 8 = dbinom(14, 22, 0.7) = 0.1423. This is the value of the density function at 14. The probability of the event of less than or equal to 13 purines equals the value of the distribution function at value 13, that is P(X ≤ 13) = pbinom(13, 22, 0.7) = 0.1865. The probability of strictly more than 10 purines is P(X ≥ 11) = 22 k=11 P(S 22 = k) = sum(dbinom(11 : 22, 22, 0.7)) = 0.9860. The binomial density function can be plotted by: 34 CHAPTER 3. IMPORTANT DISTRIBUTIONS 0 5 10 15 20 0 . 0 0 0 . 0 5 0 . 1 0 0 . 1 5 x f ( x ) Figure 3.1: Binomial probabilities with n = 22 and p = 0.7 0 5 10 15 20 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 x F ( x ) Figure 3.2: Binomial cumulative probabilities with n = 22 and p = 0.7. > x <- 0:22 > plot(x,dbinom(x,size=22,prob=.7),type="h") By the ﬁrst line the sequence of integers {1, 2, · · · , 22} is constructed and by the second the density function is plotted, where the argument h speciﬁes pins. From Figure 3.1 it can be observed that the largest probabilities oc- cur near the expectation 15.4. The graph in Figure 3.2 illustrates that the distribution is an increasing step function, with x on the horizontal axis and P(X ≤ x) on the vertical. A random sample of size 1000 from the binomial distribution with n = 22 and p = 0.7 can be drawn by the command rbinom(1000,22,0.7). This simulates the number of purines in 1000 microRNA’s each with purine prob- ability equal to 0.7 and length 22. 3.2 Continuous distributions The continuous distributions normal, T, F, and chi-squared will be deﬁned, explained and illustrated. 3.2. CONTINUOUS DISTRIBUTIONS 35 3.2.1 Normal distribution The normal distribution is of key importance because it is assumed for many (preprocessed) gene expression values. That is, the data values x 1 , · · · , x n are seen as realizations of a random variable X having a normal distribution. Equivalently one says that the data values are members of a normally dis- tributed population with mean µ (mu) and variance σ 2 (sigma squared). It is good custom to use Greek letters for population properties and N(µ, σ 2 ) for the normal distribution. The value of the distribution function is given by P(X ≤ x), the probability of the population to have values smaller than or equal to x. Various properties of the normal distribution are illustrated by the examples below. Example 1. To view members of the normal distribution load the TeachingDemos package and give the command vis.normal() to launch an interactive display of bell-shaped curves. These bell-shaped curves are also called normal densities. The curves are symmetric around µ and attain a unique maximum at x = µ. If x moves further away from the mean µ, then the curves moves to zero so that extreme values occur with small probability. Move the Mean and the Standard Deviation from the left to the right to explore their eﬀect on the shape of the normal distribution. In particular, when the mean µ increases, then the distribution moves to the right. If σ is small/large, then the distribution is steep/ﬂat. Example 2. Suppose that the expression values of gene CCND3 Cyclin D3 can be represented by X which is distributed as N(1.90, 0.5 2 ). From the graph of its density function in Figure 3.3, it can be observed that it is symmetric and bell-shaped around µ = 1.90. A density function may very well be seen as a histogram with arbitrarily small bars (intervals). The probability that the expression values are less then 1.4 is P(X < 1.4) = pnorm(1.4, 1.9, 0.5) = 0.1586. Figure 3.4 illustrates the value 0.16 of the distribution function at x = 1.4. It corresponds to the area of the blue colored surface below the graph of the density function in Figure 3.3. The probability that the expression values are larger than 2.4 is P(X ≥ 2.4) = 1 −pnorm(2.4, 1.9, 0.5) = 0.1586. 36 CHAPTER 3. IMPORTANT DISTRIBUTIONS 0 1 2 3 4 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 x d e n s i t y f ( x ) 1.4 P(X<=1.4) = 0.16 Figure 3.3: Graph of normal den- sity with mean 1.9 and standard deviation 0.5. 0 1 2 3 4 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 x n o r m a l d i s t r i b u t i o n F ( x ) 1.4 0.16 Figure 3.4: Graph of normal dis- tribution with mean 1.9 and stan- dard deviation 0.5. The probability that X is between 1.4 and 2.4 equals P(1.4 ≤ X ≤ 2.4) = pnorm(2.4, 1.9, 0.5) −pnorm(1.4, 1.9, 0.5) = 0.9545. The graph of the distribution function in Figure 3.4 illustrates that it is strictly increasing. The exact value for the quantile x 0.025 can be computed by > qnorm(0.025,1.9,0.5) [1] 0.920018 That is, the quantile x 0.025 = 0.920018. Hence, it holds that the probability of values smaller than 0.920018 equals 0.025, that is P(X ≤ 0.920018) = 0.025, as can be veriﬁed by pnorm(0.920018, 1.9, 0.5). When X is distributed as N(1.90, 0.5 2 ), then the population mean is 1.9 and the population standard deviation 0.5. To verify this we draw a random sample of size 1000 from this population by > x <- rnorm(1000,1.9,0.5) The estimate mean(x)=1.8862 and sd(x)=0.5071 are close to their popula- tion values µ = 1.9 and σ = 0.5. 2 2 Use the function round to print the mean in a desired number a decimal places. 3.2. CONTINUOUS DISTRIBUTIONS 37 For X distributed as N(µ, σ 2 ), it holds that (X−µ)/σ = Z is distributed as N(0, 1). Thus by subtracting µ and dividing the result with σ any normally distributed variable can be standardized into a standard normally distributed Z having mean zero and standard deviation one. 3.2.2 Chi-squared distribution The chi-squared distribution plays an important role in testing hypotheses about frequencies, see Chapter 4. To deﬁne it, let {Z 1 , · · · , Z m } be indepen- dent and standard normally distributed random variables. Then the sum of squares χ 2 m = Z 2 1 +· · · + Z 2 m = m i=1 Z 2 i , is the so-called chi-squared distributed (random) variable with m degrees of freedom. Example 1. To view various members of the χ 2 distribution load the TeachingDemos package. Use the command vis.gamma() to open an inter- active display of various distributions. Click on ”Visualizing the gamma”, ”Visualizing the Chi-squared”, and adapt ”Xmax”. Move the ”Shape” but- ton to the right to increase the degrees of freedom. Observe that the graphs of chi-squared densities change from heavily skew to the right into more bell- shaped normal as the degrees of freedom increases. Example 2. Let’s consider the chi-squared variable with 5 degrees of freedom; χ 2 5 = Z 2 1 + · · · + Z 2 5 . To compute the probability of values smaller than eight we use the function pchisq, as follows. P _ χ 2 5 ≤ 8 _ = pchisq(8, 5) = 0.8437644. This yields the value of the distribution function at x = 8 (see Figure 3.6). This value corresponds to the area of the blue colored surface below the graph of the density function in Figure 3.5. Often we are interested in the value for the quantile x 0.025 , where P(χ 2 5 ≤ x 0.025 ) = 0.025. 3 Such can be computed by 3 If the distribution function is strictly increasing, then there exists an exact and unique solution for the quantiles. 38 CHAPTER 3. IMPORTANT DISTRIBUTIONS 0 5 10 15 20 25 0 . 0 0 0 . 0 5 0 . 1 0 0 . 1 5 C h i S q u a r e d D e n s i t y f ( x ) 8 area=0.84 Figure 3.5: χ 2 5 -density. 0 5 10 15 20 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 C h i S q u a r e d D i s t r i b u t i o n F ( x ) 8 0 . 8 4 x Figure 3.6: χ 2 5 distribution. > qchisq(0.025, 5, lower.tail=TRUE) [1] 0.8312 Example 3. The chi-squared distribution is frequently used as a so-called goodness of ﬁt measure. With respect to the Golub et. al. (1999) data we may hypothesize that the expression values of gene CCND3 Cyclin D3 for the ALL patients are distributed as N(1.90, 0.50 2 ). If this indeed holds, then the sum of squared standardized values equals their number and the probability of larger values is about 1/2. In particular, let x 1 , · · · , x 27 be the gene expression values. Then the standardized values are z i = (x i −1.90)/0.50 and their sum of squares 27 1 z 2 i = 25.03312. The probability of larger values is P (χ 2 27 ≥ 25.03312) = 0.5726, which indicates that this normal distribution ﬁts the data well. Hence, it is likely that the speciﬁed normal distribution is indeed correct. Using R the computations are as follows. library(multtest); data(golub) gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) x <- golub[1042,gol.fac=="ALL"] z <- (x-1.90)/0.50 sum(z^2) pchisq(sum(z^2),27, lower.tail=FALSE) 3.2. CONTINUOUS DISTRIBUTIONS 39 3.2.3 T-Distribution The T-distribution has many useful applications for testing hypotheses about means of gene expression values, in particular when the sample size is lower than thirty. If the data are normally distributed, then the values of n(x − µ)/s follow a T-distribution with n−1 degrees of freedom. The T-distribution is approximately equal to the normal distribution when the sample size is thirty. −4 −2 0 2 4 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 x−axis D e n s i t y f ( x ) Figure 3.7: Density of T 10 distri- bution. −4 −2 0 2 4 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 x−axis D i s t r i b u t i o n F ( x ) Figure 3.8: Distribution function of T 10 . Example 1. Load the TeachingDemos and give vis.t() to explore a vi- sualization of the T-distribution. Click on ”Show Normal Distribution” and increase the number of degrees of freedom to verify that df equal to thirty is suﬃcient for the normal approximation to be quite precise. Example 2. A quick NCBI scan makes it reasonable to assume that the gene Gdf5 has no direct relation with leukemia. For this reason we take µ = 0. The expression values of this gene are collected in row 2058 of the golub data. To compute the sample t-value n(x −µ)/s use n <- 11 x <- golub[2058, gol.fac=="AML"] 40 CHAPTER 3. IMPORTANT DISTRIBUTIONS t.value <- sqrt(n)*(mean(x)-0)/sd(x) t.value [1] 1.236324 From the above we know that this has a T 10 distribution. The probability that T 10 is greater than 1.236324 can be computed, as follows. P(T 10 ≥ 1.236324) = 1 −P(T 10 ≤ 1.236324) = 1 −pt(1.236324, 10) = 0.12. This probability corresponds to the area of the blue colored surface below of the graph of the density function in Figure 3.7. The T distribution function with ten degrees of freedom is illustrated in Figure 3.8. The probability that the random variable T 10 is between -2 and 2 equals P(−2 ≤ T 11 ≤ 2) = pt(2, 10) −pt(−2, 10) = 0.926612. The 2.5% quantile can be computed by qt(0.025,n-1)=-2.228139. 3.2.4 F-Distribution The F-distribution is important for testing the equality of two variances. It can be shown that the ratio of variances from two independent sets of nor- mally distributed random variables follows an F-distribution. More speciﬁ- cally, if the two population variances are equal (σ 2 1 = σ 2 2 ), then s 2 1 /s 2 2 follows an F-distribution with n 1 − 1, n 2 − 1 degrees of freedom, where s 2 1 is the variance of the ﬁrst set, s 2 2 that of the second, and n 1 is the number of ob- servations in the ﬁrst and n 2 in the second. 4 Example 1. For equal population variances the probability is large that that the ratio of sample variances is near one. With respect to the Golub et. al. (1999) data it is easy to compute the ratio of the variances of the expression values of gene CCND3 Cyclin D3 for the ALL patients and the AML patients. > var(golub[1042,gol.fac=="ALL"])/var(golub[1042,gol.fac=="AML"]) [1] 0.7116441 4 It is more correct to deﬁne S 2 1 /S 2 2 for certain random variables S 2 1 and S 2 2 , we shall , however, not border. 3.2. CONTINUOUS DISTRIBUTIONS 41 0 2 4 6 8 10 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 F d e n s i t y f ( x ) x 0.71 0.23 Figure 3.9: Density of F 26,10 . 0 2 4 6 8 10 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 F D i s t r i b u t i o n F ( x ) 0.23 0.71 Figure 3.10: Distribution of F 26,10 . Since n 1 = 27 and n 2 = 11 this ratio is a realization of the F 26,10 distribution. Then, the probability that the ratio attains values smaller than 0.7116441 is P(X ≤ 0.7116441) = pf(0.7116441, 26, 10) = 0.2326147. Figure 3.9 illustrates that this value corresponds to the area of the blue col- ored surface below the graph of the density function. Figure 3.10 gives the distribution function. To ﬁnd the quantile x 0.025 use qf(.025,26,10)=0.3861673. This subject is taken further in Section 4.1.5. 3.2.5 Plotting a density function 5 A convenient manner to plot a density function in by using the correspond- ing built-in-function. For instance to plot the bell-shaped density from the normally distributed variable use the function dnorm, as follows. > f<-function(x){dnorm(x,1.9,0.5)} > plot(f,0,4,xlab="x-axis",ylab="density f(x)") 5 This subsection is solemly on plotting and can be skipped without loss of continuity. 42 CHAPTER 3. IMPORTANT DISTRIBUTIONS This produces the graph of the density function in Figure 3.3. The speciﬁca- tion 0,4 deﬁnes the interval on the horizontal axis over which f is plotted. The vertical axis is adapted automatically. We can give the surface under f running x from 0 to 1.4 a nice blue color by using the following. plot(f,0,4,xlab="x-axis",ylab="density f(x)") x<-seq(0,1.4,0.01) polygon(c(0,x,1.4), c(0,f(x),0), col="lightblue") The basic idea of plotting is to start with a plot and next to add colors, text, arrows, etc. In particular, the command polygon is used to give the surface below the graph the color "lightblue". The polygon (surface enclosed by many angles) is deﬁned by the sequence of points deﬁned as x and f(x). 3.3 Overview and concluding remarks For practical computations R has built-in-functions for the binomial, normal, t, F, χ 2 -distributions, where d stands for density, p for (cumulative) prob- ability distribution, q for quantiles, and r for drawing random samples, see Table 3.2. The density, expectation, and variance of most the distributions in this chapter are summarized in Table 3.3. Table 3.2: Built-in-functions for random variables used in this chapter. para- random Distribution meters density distribution quantiles sampling Bin n, p dbinom(x, n, p) pbinom(x, n, p) qbinom(α, n, p) rbinom(10, n, p) Normal µ, σ dnorm(x, µ, σ) pnorm(x, µ, σ) qnorm (α, µ, σ) rnorm(10, µ, σ) Chi-squared m dchisq(x, m) pchisq(x, m) qchisq(α, m) rchisq(10, m) T m dt(x, m) pt(x, m) qt(α, m) rt(10, m) F m,n df(x, m, n) pf(x, m, n) qf(α, m, n) rf(10, m, n) Although for a ﬁrst introduction the above distributions are without doubt among the most important, there are several additional distributions available such as the Poisson, Gamma, beta, or Dirichlet. Obviously, these can also be programmed by yourself. The freeware encyclopedia wikipedia of- ten gives a useful ﬁrst, though technical, orientation. Note that a distribution acts as a population from which a sample can be drawn. Hence, distributions 3.4. EXERCISES 43 can be seen as models of data generating procedures. For a more thorough treatment of distribution we refer the reader to Bain & Engelhardt (1992), Johnson et al. (1992), and Miller & Miller (1999). Table 3.3: Density, mean, and variance of distributions used in this chapter. Distribution parameters density expectation variance Binomial n, p n! k!(n−k)! p k (1 −p) n−k np np(1 −p) Normal µ, σ 1 σ exp(− 1 2 ( x−µ σ ) 2 ) µ σ 2 Chi-squared df=m m 2m 3.4 Exercises It is importance to obtain some routine with the computation of probabilities and quantiles. 1. Binomial Let X be binomially distributed with n = 60 and p = 0.4. Compute the following. (a) P(X = 24), P(X ≤ 24), and P(X ≥ 30). (b) P(20 ≤ X ≤ 30), P(20 ≤ X). (c) P(20 ≤ X or X ≥ 40), and P(20 ≤ X and X ≥ 10). (d) Compute the mean and standard deviation of X. (e) The quantiles x 0.025 , x 0.5 , and x 0.975 . 2. Standard Normal. Compute the following probabilities and quantiles. (a) P(1.6 < Z < 2.3). (b) P(Z < 1.64). (c) P(−1.64 < Z < −1.02). (d) P(0 < Z < 1.96). (e) P(−1.96 < Z < 1.96). (f) The quantiles z 0.025 , z 0.05 , z 0.5 , z 0.95 , and z 0.975 . 3. Normal. Compute for X distributed as N(10, 2) the following proba- bilities and quantiles. 44 CHAPTER 3. IMPORTANT DISTRIBUTIONS (a) P(X < 12). (b) P(X > 8). (c) P(9 < X < 10, 5). (d) The quantiles x 0.025 , x 0.5 , and x 0.975 . 4. T-distribution. Verify the following computations for the T 6 distribu- tion. (a) P(T 6 < 1). (b) P(T 6 > 2). (c) P(−1 < T 6 < 1). (d) P(−2 < T 6 < −2). (e) The quantiles t 0.025 , t 0.5 , and t 0.975 . 5. F distribution. Compute the following probabilities and quantiles for the F 8,5 distribution. (a) P(F 8,5 < 3). (b) P(F 8,5 > 4). (c) P(1 < F 8,5 < 6). (d) The quantiles f 0.025 , f 0.5 , and f 0.975 . 6. Chi-squared distribution. Compute the following for the chi-squared distribution with 10 degrees of freedom. (a) P(χ 2 10 < 3). (b) P(χ 2 10 > 4). (c) P(1 < χ 2 10 < 6). (d) The quantiles g 0.025 , g 0.5 , and g 0.975 . 7. MicroRNA. Suppose that for certain microRNA of size 20 the proba- bility of a purine is binomially distributed with probability 0.7. (a) What is the probability of 14 purines? (b) What is the probability of less than or equal to 14 purines? 3.4. EXERCISES 45 (c) What is the probability of strictly more than 10 purines? (d) By what probability is of the number of purines between 10 and 15? (e) How many purines do you expect? In other words: What is the mean of the distribution? (f) What is the standard deviation of the distribution? 8. Zyxin. The distribution of the expression values of the ALL patients on the Zyxin gene are distributed according to N(1.6, 0.4 2 ). (a) Compute the probability that the expression values are smaller than 1.2? (b) What is the probability that the expression values are between 1.2 and 2.0? (c) What is the probability that the expression values are between 0.8 and 2.4? (d) Compute the exact values for the quantiles x 0.025 and x 0.975 . (e) Use rnorm to draw a sample of size 1000 from the population and compare the sample mean and standard deviation with that of the population. 9. Some computations on Golub et al. (1999) data. (a) Take µ = 0 and compute the t-values for the ALL gene expression values. Find the three genes with largest absolute t-values. (b) Compute per gene the ratio of the variances for the ALL and the AML patients. How many are between 0.5 and 1.5? 10. Extreme value investigation. This (diﬃcult!) question aims to teach the essence of an extreme value distribution! An interesting extreme value distribution is given by Pevsner (2003, p.103). Take the maximum of a sample (with size 1000) from the standard normal distribution and repeat this 1000 times. So that you sampled 1000 maxima. Next, subtract from these maxima an and divide by bn, where an <- sqrt(2*log(n)) - 0.5*(log(log(n))+log(4*pi))*(2*log(n))^(-1/2) bn <- (2*log(n))^(-1/2) 46 CHAPTER 3. IMPORTANT DISTRIBUTIONS Now plot the density from the normalized maxima and add the extreme value function f(x) from Pevsner his book, and add the density (dnorm) from the normal distribution. What do you observe? Chapter 4 Estimation and Inference Questions that we deal with in this chapter are related to statistically testing biological hypothesis. Does the mean gene expression over ALL patients diﬀer from that over AML patients? That is, does the mean gene expression level diﬀer between experimental conditions? Is the mean gene expression diﬀerent from zero? To what extent are gene expression values normally distributed? Are there outliers among a sample of gene expression values? How can an experimental eﬀect be deﬁned? How can genes be selected with respect to an experimental eﬀect? Other important questions are: How can it be tested whether the frequencies of nucleotide sequences of two genes are diﬀerent? How can it be tested whether outliers are present in the data? What is the probability of a certain micro RNA to have more than a certain number of purines? In the foregoing chapters many population parameters were used to deﬁne families of theoretical distributions. In any research (empirical) setting the speciﬁc values of such parameters are unknown so that these must be esti- mated. Once estimates are available it becomes possible to statistically test biologically important hypotheses. The current chapter gives several basic examples of statistical testing and some of its background. Robust type of testing is brieﬂy introduced as well as an outlier test. 4.1 Statistical hypothesis testing Let µ 0 be a number representing the hypothesized population mean by a researcher on the basis of experience and knowledge from the ﬁeld. With 47 48 CHAPTER 4. ESTIMATION AND INFERENCE respect to the population mean the null hypothesis can be formulated as H 0 : µ = µ 0 and the alternative hypothesis as H 1 : µ = µ 0 . These are two statements of which the latter is the opposite of the ﬁrst: Either H 0 or H 1 is true. The alternative hypothesis is true if H 1 : µ < µ 0 or H 1 : µ > µ 0 holds true. This type of alternative hypothesis is called “two-sided”. In case H 1 : µ > µ 0 , it is called “one-sided”. Such a null hypothesis will be statistically tested against the alternative using a suitable distribution of a statistic (e.g. standardized mean). After conducting the experiment, the value of the statistic can be computed from the data. By comparing the value of the statistic with its distribution, the researcher draws a conclusion with respect to the null hypothesis: H 0 is rejected or it is not. The probability to reject H 0 , given the truth of H 0 , is called the signiﬁcance level which is generally denoted by α. We shall follow the habit in statistics to use α = 0.05, but it will be completely clear how to adapt the procedure in case other signiﬁcance levels are desired. 4.1.1 The Z-test The Z-test applies to the situation where we want to test H 0 : µ = µ 0 against H 1 : µ = µ 0 and the standard deviation σ is known. Assuming that the gene expression values (x 1 , · · · , x n ) are from a normal distribution we compute the standardized value z = n(x − µ 0 )/σ. Next we deﬁne the so-called p- value as the standard normal probability of Z attaining values being more extreme than |z|, that is occurring to the left of −|z| or to the right of |z|. 1 Accordingly, the p-value equals P(Z ≤ −|z|) + P(Z ≥ |z|) = 2 · P(Z ≤ −|z|). The conclusion from the test is now as follows: If the p-value is larger than the signiﬁcance level α, then H 0 is not rejected and if it is smaller than the signiﬁcance level, then H 0 is rejected. Example 1. To illustrate the Z-test we shall concentrate on the Gdf5 gene from the Golub et al. (1999) data 2 . The corresponding expression values are contained in row 2058. A quick search through the NCBI site 1 Recall from a calculus course that | −2| = 2 and |2| = 2. 2 We will work with golub throughout this chapter, so it is essential to load these data and to deﬁne the factor gol.fac. 4.1. STATISTICAL HYPOTHESIS TESTING 49 makes it likely that this gene is not directly related to leukemia. Hence, we may hypothesize that the population mean of the ALL expression values equals zero. Accordingly, we test H 0 : µ = 0 against H 1 : µ = 0. For the sake of illustration we shall pretend that the standard deviation σ is known to be equal to 0.25. The z-value (=0.001116211) can be computed as follows. data(golub, package = "multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) sigma <- 0.25; n <- 27; mu0 <- 0 x <- golub[2058,gol.fac=="ALL"] z.value <- sqrt(n)*(mean(x) - mu0)/sigma The p-value can now be computed as follows. > 2*pnorm(-abs(z.value),0,1) [1] 0.9991094 Since it is clearly larger than 0.05, we conclude that the null hypothesis of mean equal to zero is not rejected (accepted). Note that the above procedure implies rejection of the null hypothesis when z is highly negative or highly positive. More precisely, if z falls in the region (−∞, z 0.025 ] or [z 0.975 , ∞), then H 0 is rejected. For this reason these intervals are called “rejection regions”. If z falls in the interval (z 0.025 , z 0.975 ), then H 0 is not rejected and consequently this region is called ”acceptance region”. The situation is illustrated in Figure 4.1. The interval (z 0.025 , z 0.975 ) is often named “conﬁdence interval”, because if the null hypothesis is true, then we are 95% conﬁdent that the observed z-value falls in it. It is custom to rework the conﬁdence interval into an interval with respect to µ (Samuels & Witmer, 2003, p. 186). In particular, the 95% conﬁdence interval for the population mean µ is _ x + z 0.025 σ n , x + z 0.975 σ n _ . (4.1) That is, we are 95% certain 3 that the true mean falls in the conﬁdence inter- val. Such an interval is standard output of statistical software. 3 If we would repeat the procedure suﬃciently often 50 CHAPTER 4. ESTIMATION AND INFERENCE −4 −2 0 2 4 0 . 0 0 . 1 0 . 2 0 . 3 0 . 4 n o r m a l d e n s i t y z rejection area α 2 rejection area α 2 acceptance area 1 −α Figure 4.1: Acceptance and rejection regions of the Z-test. Example 2. Using the data from Example 1, the 95% conﬁdence interval given by Equation 4.1 can be computed as follows. 4 > mean(x)+qnorm(c(0.025),0,1)*sigma/sqrt(n) [1] -0.0942451 > mean(x)+qnorm(c(0.975),0,1)*sigma/sqrt(n) [1] 0.09435251 Hence, the rounded estimated 95% conﬁdence interval is (−0.094, 0.094). Since µ 0 = 0 falls within this interval, H 0 is not rejected. It is instructive and convenient to run the Z-test from the TeachingDemos package, as follows. 4 These computations only work together with those of Example 1, especially the deﬁ- nition of x. 4.1. STATISTICAL HYPOTHESIS TESTING 51 > library(TeachingDemos) > z.test(x,mu=0,sd=0.25) One Sample z-test data: x z = 0.0011, n = 27.000, Std. Dev. = 0.250, Std. Dev. of the sample mean = 0.048, p-value = 0.9991 alternative hypothesis: true mean is not equal to 0 95 percent confidence interval: -0.09424511 0.09435251 sample estimates: mean of x 5.37037e-05 From the z-value, the p-value, and the conﬁdence interval, the conclusion is not to reject the null-hypothesis of mean equal to zero. This illustrates that testing by either of these procedures yields equivalent conclusions. Example 3. To develop intuition with respect to conﬁdence intervals load the package TeachingDemos and give the following command. > ci.examp(mean.sim =0, sd = 1, n = 25, reps = 100, + method = "z", lower.conf=0.025, upper.conf=0.975) Then 100 samples of size 25 from the N(0, 1) distribution are drawn and for each of these the conﬁdence interval for the population mean is computed and represented as a line segment. Apart from sampling ﬂuctuations, the conﬁdence level corresponds to the percentage of intervals containing the true mean (colored in black) and that the signiﬁcance level corresponds to intervals not containing it (colored in red or blue). 4.1.2 One Sample t-Test Indeed, in almost all research situations with respect to gene expression val- ues, the population standard deviation σ is unknown so that the above test is not applicable. In such cases t-tests are very useful for testing H 0 : µ = µ 0 against H 1 : µ = µ 0 . The test is based on the t-value deﬁned by t = 52 CHAPTER 4. ESTIMATION AND INFERENCE n(x − µ 0 )/s. The corresponding p-value is deﬁned by 2 · P(T n−1 ≤ −|t|). Similar to the above, H 0 is not rejected if the p-value is larger than the signif- icance level and H 0 is rejected if the p-value is smaller than the signiﬁcance level. Equivalently, if t falls in the acceptance region (t 0.025,n−1 , t 0.975,n−1 ), then H 0 is not rejected and otherwise it is. For n = 6 the acceptance and rejection regions are illustrated in Figure 4.2. The 95% conﬁdence interval for the population mean is given by (x+t 0.025 · s/ n, x+t 0.975 · s/ n), where the expression s/ n gives the so-called “standard error of the mean”. −4 −2 0 2 4 0 . 0 0 . 1 0 . 2 0 . 3 x−axis T d e n s i t y rejection region α 2 rejection region α 2 acceptance region t 0.025 t 0.975 Figure 4.2: Acceptance and rejection regions of the T 5 -test. Example 1. Let’s test H 0 : µ = 0 against H 1 : µ = 0 for the ALL population mean of the Gdf5 gene expressions. The latter are collected in row 2058 of the golub data. The t-value is computed as follows. 4.1. STATISTICAL HYPOTHESIS TESTING 53 > x <- golub[2058,gol.fac=="ALL"]; mu0 <- 0; n <- 27 > t.value<-sqrt(n)*(mean(x) - mu0)/sd(x) > t.value [1] 0.001076867 The corresponding p-value can be computed by 2 · P(T 26 ≤ −0.0010) = 2 ∗ pt(−0.0010, 26) = 0.9991 > α, so that the conclusion is not to reject the null hypothesis of mean equal to zero. To see whether the observed t-value belongs to the 95% conﬁdence inter- val, we compute (t 0.025,26 , t 0.975,26 ) = (qt(0.025, n −1), qt(0.975, n −1)) = (−2.055, 2.055). Since this interval does contain the t-value, we do not reject the hypothesis that µ equals zero. The left boundary of the 95% conﬁdence interval for the population mean can be computed, as follows. > mean(x)+qt(0.025,26)*sd(x)/sqrt(n) [1] -0.1024562 The 95% conﬁdence interval equals (−0.1025, 0.1025). Since it contains zero, we do not reject the null-hypothesis. In daily practice it is much more convenient to use the built-in-function t.test. We illustrate it with the current testing problem. > t.test(x,mu=0) One Sample t-test data: x t = 0.0011, df = 26, p-value = 0.9991 alternative hypothesis: true mean is not equal to 0 95 percent confidence interval: -0.1024562 0.1025636 sample estimates: mean of x 5.37037e-05 54 CHAPTER 4. ESTIMATION AND INFERENCE This yields by one command line the observed t-value, the p-value, and the 95% conﬁdence interval for µ 0 . In the previous example the test is two-sided because H 1 holds true if µ < µ 0 or µ > µ 0 . If, however, the researcher desires to test H 0 : µ = µ 0 against H 1 : µ > µ 0 , then the alternative hypothesis is one-sided and this makes the procedure slightly diﬀerent: H 0 is accepted if P(T n ≥ t) > α and it is rejected if P(T n ≥ t) < α. We shall illustrate this by a variant of the previous example. Example 2. In Chapter 2 a box-and-whiskers plot revealed that the ALL gene expression values of CCND3 Cyclin D3 are positive. Hence, we test H 0 : µ = 0 against H 1 : µ > 0 by the built-in-function t-test. Recall that the corresponding gene expression values are collected in row 1042 of the golub data matrix (load it if necessary). > t.test(golub[1042,gol.fac=="ALL"],mu=0, alternative = c("greater")) One Sample t-test data: golub[1042, gol.fac == "ALL"] t = 20.0599, df = 26, p-value < 2.2e-16 alternative hypothesis: true mean is greater than 0 95 percent confidence interval: 1.732853 Inf sample estimates: mean of x 1.893883 The large t-value indicates that, relative to its standard error, the mean dif- fers largely from zero. Accordingly, the p-value is very close to zero, so that the conclusion is to reject H 0 . 4.1.3 Two-sample t-test with unequal variances Suppose that gene expression data from two groups of patients (experimen- tal conditions) are available and that the hypothesis is about the diﬀerence between the population means µ 1 and µ 2 . In particular, H 0 : µ 1 = µ 2 is to 4.1. STATISTICAL HYPOTHESIS TESTING 55 be tested against H 1 : µ 1 = µ 2 . These hypotheses can also be formulated as H 0 : µ 1 − µ 2 = 0 and H 1 : µ 1 − µ 2 = 0. Suppose that gene expression data from the ﬁrst group are given by {x 1 , · · · x n } and that of the second by {y 1 , · · · , y m }. Let x be the mean of the ﬁrst and y that of the second, and s 2 1 the variance of the ﬁrst and s 2 2 that of the second. Then the t-statistic can be formulated as t = (x −y) −(µ 1 −µ 2 ) _ s 2 1 /n + s 2 2 /m . (4.2) The decision procedure with respect to the null-hypothesis is completely sim- ilar to the above tests. Note that the t-value is large if the diﬀerence between x and y is large 5 , the standard deviations s 1 and s 2 are small, and the sample sizes are large. This test is known as the Welch two-sample t-test (Lehmann, 1999). Example 1. Golub et al. (1999) argue that gene CCND3 Cyclin D3 plays an important role with respect to discriminating ALL from AML patients. The boxplot in Figure 2.4 suggests that the ALL population mean diﬀers from that of AML. The null hypothesis of equal means can be tested by the func- tion t.test and the appropriate factor and speciﬁcation var.equal=FALSE. > t.test(golub[1042,] ~ gol.fac, var.equal=FALSE) Welch Two Sample t-test data: golub[1042, ] by gol.fac t = 6.3186, df = 16.118, p-value = 9.87e-06 alternative hypothesis: true difference in means is not equal to 0 95 percent confidence interval: 0.8363826 1.6802008 sample estimates: mean in group ALL mean in group AML 1.8938826 0.6355909 The t-value is quite large, indicating that the two means x and y diﬀer largely from zero relative to the corresponding standard error (denominator in Equa- tion 4.2). Since the p-value is extremely small, the conclusion is to reject the null-hypothesis of equal means. The data provide strong evidence that the 5 Assuming µ 1 −µ 2 = 0. 56 CHAPTER 4. ESTIMATION AND INFERENCE population means do diﬀer. When the ﬁrst group is an experimental group and the second a control group, then µ 1 −µ 2 is the experimental eﬀect in the population and x−y that in the sample. The t-value is the experimental eﬀect in the sample relative to the standard error. The size of the eﬀect is measured by the p-value in the sense that it is smaller for larger eﬀects. If the two population variances are equal, then the testing procedure simpliﬁes considerably. This is the subject of the next paragraph. 4.1.4 Two sample t-test with equal variances Suppose exactly the same setting as in the previous paragraph, but now the variances σ 2 1 and σ 2 2 for the two groups are known to be equal. To test H 0 : µ 1 = µ 2 against H 1 : µ 1 = µ 2 , there is a t-test which is based on the so-called pooled sample variance s 2 p . The latter is deﬁned by the following weighted sum of the sample variances s 2 1 and s 2 2 , namely s 2 p = (n −1)s 2 1 + (m−1)s 2 2 n + m−2 . Then the t-value can be formulated as t = x −y −(µ 1 −µ 2 ) s p _ 1 n + 1 m . Example 1. The null hypothesis for gene CCND3 Cyclin D3 that the mean of the ALL diﬀers from that of AML patients can be tested by the two-sample t-test using the speciﬁcation var.equal=TRUE. > t.test(golub[1042,] ~ gol.fac, var.equal = TRUE) Two Sample t-test data: golub[1042, ] by gol.fac t = 6.7983, df = 36, p-value = 6.046e-08 alternative hypothesis: true difference in means is not equal to 0 95 percent confidence interval: 0.8829143 1.6336690 4.1. STATISTICAL HYPOTHESIS TESTING 57 sample estimates: mean in group ALL mean in group AML 1.8938826 0.6355909 From the p-value 6.046 · 10 −8 , the conclusion is to reject the null hypothesis of equal population means. Note that the p-value is slightly smaller than that of the previous test. In case of any uncertainty about the validity of the assumption of equal population variances, one may want to test this. 4.1.5 F-test on equal variances The assumption of the above t-test it that the two population variances are equal. Such an assumption can serve as a null hypothesis. That is, we desire to test H 0 : σ 2 1 = σ 2 2 against H 0 : σ 2 1 = σ 2 2 . This can be accomplished by the so-called F-test, as follows. From the sample variances s 2 1 and s 2 2 , the f-value f = s 2 1 /s 2 2 can be computed, which is F n 1 −1,n 2 −1 distributed with n 1 −1 and n 2 −1 degrees of freedom. If P(F n 1 −1,n 2 −1 < f) ≥ α/2 for f < 1 or P(F n 1 −1,n 2 −1 > f) ≥ α/2 for f > 1, then H 0 is not rejected and otherwise it is rejected. Example 1. The null hypothesis for gene CCND3 Cyclin D3 that the variance of the ALL patients equals that of the AML patients can be tested by the built-in-function var.test, as follows. > var.test(golub[1042,] ~ gol.fac) F test to compare two variances data: golub[1042, ] by gol.fac F = 0.7116, num df = 26, denom df = 10, p-value = 0.4652 alternative hypothesis: true ratio of variances is not equal to 1 95 percent confidence interval: 0.2127735 1.8428387 sample estimates: ratio of variances 0.7116441 58 CHAPTER 4. ESTIMATION AND INFERENCE From the p-value 0.4652, the null-hypothesis of equal variances is not re- jected. 4.1.6 Binomial test Suppose that for a certain micro RNA a researcher wants to test the hy- pothesis that the probability of a purine equals a certain value p 0 . However, another researcher has reason to believe that this probability is larger. In such a setting we want to test the null-hypothesis H 0 : p = p 0 against the one-sided alternative hypothesis H 1 : p > p 0 . Suppose that sequencing re- veals that the micro RNA has k purines out of a total n. Assuming that the binomial distribution holds, the null-hypothesis can be tested by computing the p-value P(X ≥ k). If it is larger than the signiﬁcance level α = 0.05, then H 0 is not rejected and otherwise it is. Example 1. A micro RNA of length 22 contains 18 purines. The null hypothesis H 0 : p = 0.7 is to be tested against the one-sided H 1 : p > 0.7. From P(X ≥ 18) = 1 −pbinom(17, 22, 0.7) = 0.1645 ≥ 0.05 = α, the conclusion follows not to reject the null-hypothesis. This test can also be conducted by the function binom.test as follows. > binom.test(18, 22, p = 0.7, alternative = c("greater"), + conf.level = 0.95) Exact binomial test data: 18 and 22 number of successes = 18, number of trials = 22, p-value = 0.1645 alternative hypothesis: true probability of success is greater than 0.7 95 percent confidence interval: 0.6309089 1.0000000 sample estimates: probability of success 0.8181818 The p-value 0.1645 is larger than the signiﬁcance level 0.05, so that the null hypothesis is not rejected. 4.1. STATISTICAL HYPOTHESIS TESTING 59 4.1.7 Chi-squared test It often happens that we want to test a hypothesis with respect to more than one probability. That is, the H 0 : (π 1 , · · · , π m ) = (p 1 , · · · , p m ) against H 1 : 1 , · · · , π m ) = (p 1 , · · · , p m ), where p 1 to p m are given numbers corresponding to the hypothesis of a researcher. By multiplying the probabilities with the total number of observations we obtain the expected number of observations (e i = n · p i ). Now we can compute the statistic q = m i=1 (o i −e i ) 2 /e i , where o i is the i-th observed and e i the i-th expected frequency. This statistic is chi-squared (χ 2 m−1 ) distributed with m− 1 degrees of freedom. The p-value of the chi-squared test is deﬁned as P(χ 2 m−1 ≥ q). If it is larger than the signiﬁcance level, then the null hypothesis is not rejected, and otherwise it is. 0 5 10 15 20 25 0 . 0 0 0 . 0 5 0 . 1 0 0 . 1 5 0 . 2 0 0 . 2 5 C h i S q u a r e d D e n s i t y f ( x ) rejection region acceptance region q 7.8 Figure 4.3: Rejection region of χ 2 3 -test. 60 CHAPTER 4. ESTIMATION AND INFERENCE Example 1. Suppose we want to test the hypothesis that the nucleotides of Zyxin have equal probability. Let the probability of {A, C, G, T} to occur in the sequence be given by (π 1 , π 2 , π 3 , π 4 ). Then the null hypothesis to be tested is (π 1 , π 2 , π 3 , π 4 ) = (1/4, 1/4, 1/4, 1/4). In particular, for the sequence ”X94991.1” from Table 1.1 the total number of nucleotides is n = 2166, so that the expected frequencies e i are equal to 2166/4 = 541.5. Then, the q-value equals 4 i=1 (o i −e i ) 2 /e i = (410 −541.5) 2 541.5 + (789 −541.5) 2 541.5 + (573 −541.5) 2 541.5 + (394 −541.5) 2 541.5 = 187.0674 Since, P(χ 2 [3] ≥ 187.0674) is close to zero, the null hypothesis is clearly rejected. The nucleotides of Zyxin do not occur with equal probability. A more direct manner to perform the test is by using the built-in-function chisq.test, as follows. > library(ape) > zyxinfreq <- table(read.GenBank(c("X94991.1"),as.character=TRUE)) > chisq.test(zyxinfreq) Chi-squared test for given probabilities data: zyxinfreq X-squared = 187.0674, df = 3, p-value < 2.2e-16 The package ape is loaded, the Zyxin sequence "X94991.1" is downloaded, and the frequency table is constructed. The observed frequencies are given as input to chisq.test which has equal probabilities as the default option. The q-value equals X-squared and the degrees of freedom df = 3. From the corresponding p-value, the conclusion is to reject the null hypothesis of equal probabilities. The testing situation is illustrated in Figure 4.3, where the red colored surface corresponds to the rejection region (7.81, ∞). Remember from the previous chapter that the left bound of this rejection interval can by found by qchisq(0.95, 3). The observed q = 187.0674 obviously falls far into the right hand side of the rejection region, so that the corresponding p-value is very close to zero. Example 2. In the year 1866 Mendel observed in large number of exper- iments frequencies of characteristics of diﬀerent kinds of seed and their oﬀ- spring. In particular, this yielded the frequencies 5474, 1850 the seed shape 4.1. STATISTICAL HYPOTHESIS TESTING 61 of ornamental sweet peas. A crossing of B and b yields oﬀ spring BB, Bb and bb with probability 0.25, 0.50, 0.25. Since Mendel could not distinguish Bb from BB, his observations theoretically occur with probability 0.75 (BB and Bb) and 0.25 (bb). To test the null hypothesis H 0 : (π 1 , π 2 ) = (0.75, 0.25) against H 1 : (π 1 , π 2 ) = (0.75, 0.25), we use the chi-squared test 6 , as follows. > pi <- c(0.75,0.25) > x <-c(5474, 1850) > chisq.test(x, p=pi) Chi-squared test for given probabilities data: x X-squared = 0.2629, df = 1, p-value = 0.6081 From the p-value 0.6081, we do not reject the null hypothesis. To further illustrate the great ﬂexibility of the chi-squared test another example is given. Example 3. Given certain expression values for a healthy control group and an experimental group with a disease, we may deﬁne a certain cut oﬀ value and classify e.g. smaller values to be healthy and larger ones to be infected. In such a manner cut-oﬀ values can serve as a diagnostic instru- ment. The classiﬁcation yields true positives (correctly predicted disease), false positives (incorrectly predicted disease), true negatives (correctly pre- dicted healthy) and false negatives (incorrectly predicted healty). For the sake of illustration suppose that among twenty patients there are 5 true pos- itives (tp), 5 false positives (fp), 5 true negatives (tn), and 5 false negatives (fn). These frequencies can be put is a two-by-two table giving the frequen- cies on two random variables: the true state of the persons and the predicted state of the persons (by the cut oﬀ value). In the worst case the prediction by the cut-oﬀ value is independent of the disease state of the patient. The null hypothesis of independence, can be tested by a chi-square test, as follows. > dat <- matrix(c(5,5,5,5),2,byrow=TRUE) > chisq.test(dat) 6 For the sake of clarity the code is somewhat unelegant in using the symbol pi, the constant representing the ratio of a circle’s circumference to its diameter. 62 CHAPTER 4. ESTIMATION AND INFERENCE Pearson’s Chi-squared test with Yates’ continuity correction data: dat X-squared = 0.2, df = 1, p-value = 0.6547 Since the p-value is larger than the signiﬁcance level, the null hypothesis of independence is not rejected. Suppose that for another cutoﬀ value we obtain 8 true positives (tp), 2 false positives (fp), 8 true negatives (tn), and 2 false negatives (fn). Then testing independence yields the following. > dat <- matrix(c(8,2,2,8),2,byrow=TRUE) > chisq.test(dat) Pearson’s Chi-squared test with Yates’ continuity correction data: dat X-squared = 5, df = 1, p-value = 0.02535 Since the p-value is smaller than the signiﬁcance level, the null hypothesis of independence is rejected. signiﬁcant non-signiﬁcant genes genes Chromosome 1 100 2000 genome 300 6000 Example 4. A related and frequently applied test in Bioinformatics is the Fisher exact test. In a two by two table with frequencies f 11 , f 22 , (f 12 , and f 21 ), this test is based on the so-called odds ratio f 11 f 22 /(f 12 f 21 ). Suppose that the number of signiﬁcant onco type of genes in Chromosome 1 is f 11 = 100 out of a total of f 12 = 2000 and the number of signiﬁcant genes in the whole genome is f 21 = 300 out of a total of f 22 = 6000. Then the odds ratio equals 100 · 6000/(2000 · 300) = 1 and the number of signiﬁcant oncogenes in Chromosome 1 is exactly proportional to that in the genome. The null-hypothesis of the Fisher test is that the odds ratio equals 1 and the alternative hypothesis that it diﬀers from 1. Suppose that the frequencies 4.1. STATISTICAL HYPOTHESIS TESTING 63 of signiﬁcant oncogenes for Chromosome 1 equals f 11 = 300 out of a total of f 12 = 500 and for the genome f 21 = 3000 out of f 22 = 6000. The hypothesis that the odd ratio equals one can now be tested as follows. > dat <- matrix(c(300,500,3000,6000),2,byrow=TRUE) > fisher.test(dat) Fisher’s Exact Test for Count Data data: dat p-value = 0.01912 alternative hypothesis: true odds ratio is not equal to 1 95 percent confidence interval: 1.029519 1.396922 sample estimates: odds ratio 1.199960 Since the p-value is smaller than the signiﬁcance level, the null hypothesis of odds ratio equal to one is rejected. There are more signiﬁcant oncogenes in Chromosome 1 compared to that in the genome. Other examples of the Fisher test will be given in Chapter 6. 4.1.8 Normality tests Various procedures are available to test the hypothesis that a data set is normally distributed. The Shapiro-Wilk test is based on the degree of lin- earity in a Q-Q plot (Lehmann, 1999, p.347) and the Anderson-Darling test is based on the distribution of the data (Stephens, 1986, p.372). Example 1. To test the hypothesis that the ALL gene expression values of CCND3 Cyclin D3 from Golub et al. (1999) are normally distributed, the Shapiro-Wilk test can be used as follows. > shapiro.test(golub[1042, gol.fac=="ALL"]) Shapiro-Wilk normality test 64 CHAPTER 4. ESTIMATION AND INFERENCE data: golub[1042, gol.fac == "ALL"] W = 0.947, p-value = 0.1774 Since the p-value is greater than 0.05, the conclusion is not to reject the null hypothesis that CCND3 Cyclin D3 expression values follow from a normal distribution. The Anderson-Darling test is part of the nortest package which probably needs to be installed and loaded ﬁrst. Running the test on our CCND3 Cyclin D3 gene expression values comes down to the following. > library(nortest) > ad.test(golub[1042,gol.fac=="ALL"]) Anderson-Darling normality test data: scale(golub[1042, gol.fac == "ALL"]) A = 0.5215, p-value = 0.1683 Hence, the same conclusion is drawn as from the Shapiro-Wilk test. Note that the p-values from both tests are somewhat low. This conﬁrms our obser- vation in Section 2.1.5 based on the Q-Q plot that the distribution resembles the normal. From the normality tests the conclusion is that the diﬀerences in the left tail are not large enough to reject the null-hypothesis that the CCND3 Cyclin D3 expression values are normally distributed. 4.1.9 Outliers test When gene expression values are not normally distributed, then outliers may appear with large probability. The appearance of outliers in gene expression data may inﬂuence the value of a (non-robust) statistic to a large extent. For this reason it is useful to be able to test whether a certain set of gene expression values is contaminated by an outlier or not. Accordingly, the null-hypothesis to be tested is that a set of gene expression values does not contain an outlier and the alternative is that it is contaminated with at least one outlier. Under the assumption that the data are realizations of one and the same distribution, such a hypothesis can be tested by the Grubbs (1950) test. This test is based on the statistic g = |suspect value −x|/s, where the suspect value is included for the computation of the mean x and the standard 4.1. STATISTICAL HYPOTHESIS TESTING 65 deviation s. Example 1. From Figure 2.4 we have observed that expression values of gene CCND3 Cyclin D3 may contain outliers with respect to the left tail. This can actually be tested by the function grubbs.test of the outliers package, as follows. > library(outliers) > grubbs.test(golub[1042, gol.fac=="ALL"]) Grubbs test for one outlier data: golub[1042, gol.fac == "ALL"] G = 2.9264, U = 0.6580, p-value = 0.0183 alternative hypothesis: lowest value 0.45827 is an outlier Since the p-value is smaller than 0.05, the conclusion is to reject the null- hypothesis of no outliers. In case the data are normally distributed, the probability of outliers is small. Hence, extreme outliers indicate that the data are non-normally dis- tributed with large probability. Outliers may lead to such an increase of the standard error that a true experimental eﬀect remains uncovered (false negatives). In such cases a robust test based on ranks may be preferred as a useful alternative. 4.1.10 Wilcoxon rank test In case the data are normally distributed with equal variance, the t-test is an optimal test for testing H 0 : µ 1 = µ 2 against H 1 : µ 1 = µ 2 (Lehmann, 1999). If, however, the data are not normally distributed due to skewness or otherwise heavy tails, then this optimality does not hold anymore and there is no guarantee that the signiﬁcance level of the test equals the intended level α (Lehmann, 1999). For this reason rank type of tests are developed for which on beforehand no speciﬁc distributional assumptions need to be made. In the below we shall concentrate on the two-sample Wilcoxon test because of its relevance to bioinformatics. We sustain with a brief description of the basic idea and refer the interested reader to the literature on non-parametric 66 CHAPTER 4. ESTIMATION AND INFERENCE testing (e.g. Lehmann, 2006). To broaden our view we switch from hypothe- ses about means to those about distributions. An alternative hypothesis may then be formulated as that the distribution of a ﬁrst group lays to the left of a second. To set the scene let the gene expression values of the ﬁrst group (x 1 to x m ) have distribution F and those of the second group (y 1 to y n ) distribution G. The null hypothesis is that both distributions are equal (H 0 : F = G) and the alternative that these are not. For example that the x’s are smaller (or larger) than the y’s. By the two-sample Wilcoxon test the data x 1 , · · · , x m , y 1 , · · · , y n are ranked and the rank numbers of the x’s are summed to form the statistic W after a certain correction (Lehmann, 2006). The idea is that if the ranks of x’s are smaller than those of the y’s, then the sum is small. The distribution of the sum of ranks is known so that a p-value can be computed on the basis of which the null hypothesis is rejected if it is smaller than the signiﬁcance level α. Example 1. The null hypothesis that the expression values for gene CCND3 Cyclin D3 are equally distributed for the ALL patients and the AML patients can be tested by the built-in-function wilcox.test, as follows. > wilcox.test(golub[1042,] ~ gol.fac) Wilcoxon rank sum test data: golub[1042, ] by gol.fac W = 284, p-value = 6.15e-07 alternative hypothesis: true location shift is not equal to 0 Since the p-value is much smaller than α = 0.05, the conclusion is to reject the null-hypothesis of equal distributions. 4.2 Application of tests to a whole set gene expression data Various tests are applied in the above to a single vector of gene expressions. In daily practice, however, we want to analyze a set of thousands of (row) vectors with gene expression values which are collected in a matrix. Such 4.2. APPLICATIONOF TESTS TOAWHOLE SET GENE EXPRESSIONDATA67 can conveniently be accomplished by taking advantage of the fact that R stores the output of a test as an object in such a manner that we can extract information such as p-values. Recall that the smaller the p-value the larger the experimental eﬀect. Hence, by collecting p-values in a vector we can select genes with large diﬀerences between patient groups. This and testing for normality will be illustrated by two examples. Example 1. Having a data matrix with gene expression values, a ques- tion one might ask is: What is the percentage of genes that passes a normality test? Such can be computed as follows. > data(golub,package="multtest") > gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) > sh <- apply(golub[,gol.fac=="ALL"], 1, function(x) shapiro.test(x)$p.value)
> sum(sh > 0.05)/nrow(golub) * 100
[1] 58.27598
Hence, according to the Shapiro-Wilk test, 58.27% of the ALL gene ex-
pression values is normally distributed (in the sense of non-rejection). For
the AML expression values this is 60.73%. It can be concluded that about
forty percent of the genes do not pass the normality test.
Example 2. In case the gene expression data are non-normally dis-
tributed the t-test may indicate conclusions diﬀerent from those of the Wilcoxon
test. Diﬀerences between these can be investigated by collecting the p-values
from both tests and seeking for the largest diﬀerences.
> data(golub, package = "multtest");
> gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
> pt <- apply(golub, 1, function(x) t.test(x ~ gol.fac)$p.value) > pw <- apply(golub, 1, function(x) wilcox.test(x ~ gol.fac)$p.value)
> resul <- data.frame(cbind(pw,pt))
> resul[pw<0.05 & abs(pt-pw)>0.2,]
pw pt
456 0.04480288 0.2636088
1509 0.03215830 0.4427477
The p-value is extracted from the output of the t.test function and stored
in the vector pt. The logical operator & is used to select genes for which the
68 CHAPTER 4. ESTIMATION AND INFERENCE
Wilcoxon p-value is smaller than 0.05 and the absolute diﬀerence with the
p-value from the t-test is larger than 0.2. Since there are only two such genes
we can draw the reassuring conclusion that the tests give similar results.
4.3 Overview and concluding remarks
Statistical hypothesis testing consists of hypotheses, distributional assump-
tions, and decisions (conclusions). The hypotheses pertain to the outcome
of a biological experiment and are always formulated in terms of population
values of parameters. Statistically, the outcomes of experiments are seen as
realizations of random variables. The latter are assumed to have a certain
suitable distribution which is seen as a statistical model for outcomes of an
experiment. Then a statistic is formulated (e.g. a t-value) which is treated
both as a function of the random variables and as a function of the data
values. By comparing the distribution of the statistic with the value of the
statistic, the p-value is computed and compared to the level of signiﬁcance.
A large p-value indicates that the model ﬁts the data well and that the as-
sumptions as well as the null-hypothesis are correct with large probability.
However, a low p-value indicates, under the validity of the distributional as-
sumptions, that the outcome of the experiment is so unlikely that this causes
a suﬃcient amount of doubt to the researcher to reject the null hypothesis.
The quality of a test is often expressed in terms of eﬃciency, which is
usually directly related to the (asymptotic) variance of an estimator. The
relative eﬃciency is the ratio of the asymptotic variances. For Wilcoxon’s test
versus the t-test this equals .955, which means that in the optimal situation
where the (gene expression) data are normally distributed, Wilcoxon’s test
is only a little worse than the t-test. In case, however, of a few outliers or
a slightly heavier tail, the Wicoxon test can be far more eﬃcient than the
t-test (Lehmann, 1999, p.176). Eﬃciency is directly related to power; the
probability to reject a false hypothesis. The probability of drawing correct
conclusions can always be improved by increasing the sample size.
These considerations set the scene for making some recommendations,
which obviously should not be followed blindly. If gene expression data pass
a normality test, then the Welch type of t-test provides a general test with
good power properties (Ramsey, 1980; Wang, 1971). In case normality does
not hold and the sample size per group is at least least four, the Wilcoxon
4.4. EXERCISES 69
test is recommended.
Because the Wilcoxon p-values are based on ranks many of these are
equal for diﬀerent genes, so that it is less suitable for ordering in case of
small sample size. On the other hand, it is obviously questionable whether
extremely small diﬀerences in p-values produced by the t-test contribute to
biologically relevant gene discrimination. That is, extremely small diﬀerences
should not be over-interpreted.
4.4 Exercises
1. Gene CD33. Use grep to ﬁnd the index of the important gene CD33
among the list of characters golub.gnames. For each test below for-
mulate the null hypothesis, the p-value and your conclusion.
(a) Test the normality of the ALL and AML expression values.
(b) Test for the equality of variances.
(c) Test for the equality of the means by an appropriate t-test.
(d) Is the experimental eﬀect strong?
2. Gene ”MYBL2 V-myb avian myeloblastosis viral oncogene homolog-
like 2” has its expression values in row 1788.
(a) Use a boxplot to construct a hypothesis about the experimental
eﬀect.
(b) Test for the equality of means by an appropriate t-test.
3. HOXA9. Gene ”HOXA9 Homeo box A9” with expression values in row
1391, can cause leukemia (Golub et al., 1999).
(a) Test the normality of the expression values of the ALL patients.
(b) Test for the equality of means by an appropriate t-test.
4. Zyxin. On NCBI there are various cDNA clones of zyxin.
(a) Find the accession number of cDNA clone with IMAGE:3504464.
(b) Test whether the frequencies of the nucleotides are equal for each
nucleic acid.
70 CHAPTER 4. ESTIMATION AND INFERENCE
(c) Test whether the frequencies of ”X94991.1” can be predicted by
the probabilities of the cDNA sequence ”BC002323.2”.
5. Gene selection. Select the genes from the golub data with smallest
two-sample t-test values for which the ALL mean is greater than the
AML mean. Report the names of the best ten. Scan the Golub (1999)
article for genes among the ten you found and discuss their biological
function brieﬂy.
6. Antigenes. Antigenes play an important role in the development of
cancer. Order the antigenes according to their p-values from the Welch
two-sample t-test with respect to gene expression values from the ALL
and AML patients of the Golub et al. (1999) data.
7. Genetic Model. A certain genetic model predicts that four phenotypes
occur in ration 9:3:3:1. In a certain experiment the oﬀspring is observed
with frequencies 930, 330, 290, 90. Do the data conﬁrm the model?
8. Comparing two genes. Consider the gene expression values in row 790
and 66 of the Golub et al. (1999) data.
(a) Produce a boxplot for the ALL expression values and comment on
the diﬀerences. Are there outliers?
(b) Compute the mean and the median for the ALL gene expression
values for both genes. Do you observed diﬀerence between genes?
(c) Compute three measures of spread for the ALL expression values
for both genes. Do you observe diﬀerence between genes?
(d) Test by Shapiro-Wilk and Anderson-Darling the normality for the
ALL gene expression values for both genes.
9. Normality tests for gene expression values of the Golub et al. (1999)
data. Perform the Shapiro-Wilk normality test separately for the ALL
and AML gene expression values. What percentage passed the normal-
ity test separately for the ALL and the AML gene expression values?
What percentage passes both testes?
10. Two-sample tests on gene expression values of the Golub et al. (1999)
data.
4.4. EXERCISES 71
(a) Perform the two-sample Welch t-test and report the names of the
ten genes with the smallest p-values.
(b) Perform the Wilcoxon rank test and report the names of the ten
genes with the smallest p-values.
11. Biological hypotheses. Suppose that the probability to reject a biolog-
ical hypothesis by the results of a certain experiment is 0.05. Suppose
that the experiment is repeated 1000 times.
(a) How many rejections do you expect.
(b) What is the probability of less than 10 rejections?
(c) What is the probability of more than 5 rejections?
(d) What is the probability that the number of rejections is between
two and eight?
12. Programming some tests.
(a) Program the two-sample t-test with equal variances and illustrate
it with the expression values of row 1024 the of Golub et al. (1999)
data.
(b) The value of W in the two-sample Wilxoxon test equals the sum
of the ranks of Group 1 minus n(n +1)/2, where n is the number
of gene expression values in Group 1. Program this and illustrate
it with the expression values of row 1024 of Golub et al. (1999)
data.
(c) The value of W in the two-sample Wilxoxon test equals the num-
ber of values x
i
> y
j
, where x
i
, y
j
are values from Group 1 and
2, respectively. Program this and illustrate it with the expression
values of row 1024 of Golub et al. (1999) data.
72 CHAPTER 4. ESTIMATION AND INFERENCE
Chapter 5
Linear Models
We have seen that the t-test can be used to discover genes with diﬀerent
means in the population with respect to two groups of patients. In case,
however, there are three groups of patients the question arises how genes can
be selected having the largest diﬀerential expressions between group means
(experimental eﬀect)? A technique making this possible is an application of
the linear model and is called analysis of variance. It is frequently applied
bioinformatics.
The validity of the technique is based on the assumption that the gene
expression values are normally distributed and have equal variance across
groups of patients. It is of importance to investigate these assumptions be-
cause it either reassures our conﬁdence in the conclusions or it indicates that
alternative tests should be used.
In this chapter the linear model will brieﬂy be explained. The main focus,
however, is on application of the linear model for testing the hypothesis that
three or more group means are equal. Several illustrations of analyzing gene
expression data will be given. It will be explained how the assumptions about
normality and equal variances (homogeneity) can be investigated and what
alternatives can be used in case either of these does not hold. The somewhat
technical concepts of “model matrix” and “contrast matrix” are explained
because these are useful for several applications in the next chapter.
73
74 CHAPTER 5. LINEAR MODELS
5.1 Deﬁnition of linear models
Given a gene expression Y
i
, a basic form of the linear model is
Y
i
= x
i
β + ε
i
, for i = 1, · · · , n,
where Y
i
is an observable variable, x
i
a ﬁxed number, β an unknown weight,
ε
i
a unobservable error variable. The ﬁxed number x
i
follows from a sta-
tistical “design”, as we shall see. The x
i
value is part of the predictor, Y
i
the criterion, and ε
i
the error of the model. The systematical part of the
model x
i
β equals the mean of the gene expression Y
i
. The model is called
”linear” because the degree of the coeﬃcient β is one. For a linear model
to be a statistical model there must be some assumption with respect to
the distribution of the error variables. Frequently, it is assumed that the er-
ror variables ε
1
, · · · , ε
n
are independent and normally distributed with zero
mean, that is, according to N(0, σ
2
). Then the mean of Y
i
equals x
i
β and its
variance σ
2
.
Example 1. A common manner to introduce the linear model is by writing
Y
i
= β
1
+ x
i
β
2
+ ε
i
, for i = 1, · · · , n,
so that the model part represents a straight line with intercept β
1
and
slope β
2
. Given data points y
1
, · · · , y
n
and x
1
, · · · , x
n
, a best ﬁtting line
through the data can easily be computed by least squares estimation of the
intercept and slope. A nice application to explore this is by the function
put.points.demo() from the TeachingDemos package. It allows points to
be added and deleted to a plot which interactively computes estimates for
the slope and the intercept given the data. By choosing the points more or
less on a horizontal line, the slope will be near zero. By choosing the points
nearly vertical, the slope will be large. By choosing a few gross errors in the
data it can be observed that the estimates are not robust against outliers.
In order to handle gene expression data for three or more groups of pa-
tients we need to extend the model. The idea simply is to increase the
number of weights to the number of groups k, so that, we obtain the weights
β
1
, · · · , β
k
and the corresponding design values x
i1
, · · · , x
ik
. The system-
atic part of the model consists of a weighted sum of these design values:
5.1. DEFINITION OF LINEAR MODELS 75
x
i1
β
1
+· · · +x
ik
β
k
. By adding measurement error to this systematic part we
obtain the linear model
Y
i
=
k

j=1
x
ij
β
j
+ ε
i
.
The design values x
ij
for Patient i in Group j are collected in the so-called
”design” matrix denoted by X. In particular, the design value x
ij
is chosen
to be equal to 1 if Patient i belongs to Group j and zero if (s)he does not.
By this choice it becomes possible to use linear model estimation for testing
hypotheses about group means. This will be illustrated by an example.
Example 2. Suppose we have the following artiﬁcial gene expressing values
2,3,1,2, of Group 1, 8,7,9,8 of Group 2, and 11,12,13,12 of Group 3. We may
assign these to a vector y, as follows.
> y <- c(2,3,1,2, 8,7,9,8, 11,12,13,12)
Next, we construct a factor indicating to which group each expression value
belongs. In particular, the ﬁrst four belong to Group 1, the second four to
Group 2, and the third four to Group 3. We conveniently use the function
gl to deﬁne the corresponding factor.
> a <- gl(3,4)
> a
[1] 1 1 1 1 2 2 2 2 3 3 3 3
Levels: 1 2 3
The design matrix X is also called “model matrix”. It is illuminating to
print it to the screen.
> model.matrix(y ~ a - 1)
a1 a2 a3
1 1 0 0
2 1 0 0
3 1 0 0
4 1 0 0
5 0 1 0
6 0 1 0
7 0 1 0
8 0 1 0
76 CHAPTER 5. LINEAR MODELS
9 0 0 1
10 0 0 1
11 0 0 1
12 0 0 1
The notation y~a-1 represents a model equation, where -1 means to skip
the intercept or general constant.
1
In this situation, the weights (β
1
, β
2
, β
3
)
of the model specialize to the population means (µ
1
, µ
2
, µ
3
). The model for
the ﬁrst gene expression value of Group 1 is Y
1
= µ
1
+ ε
1
, for the second
expression value of Group 1 it is Y
2
= µ
1

2
, for the ﬁrst member of Group
2 it is Y
5
= µ
2
+ ε
5
, and for the ﬁrst member of Group 3 it is Y
9
= µ
3
+ ε
9
.
Recall that population means are generally estimated by sample means.
Similarly, in the current setting, estimation of the linear model comes down
to estimation of group means for which there are one-sample t-type of tests
available (see e.g. Rao & Toutenburg, 1995; Samuels & Witmer, 2003). To
illustrate this we employ the estimation function lm and ask for a summary.
> summary(lm(y ~ a - 1))
Coefficients:
Estimate Std. Error t value Pr(>|t|)
a1 2.0000 0.4082 4.899 0.000849 ***
a2 8.0000 0.4082 19.596 1.09e-08 ***
a3 12.0000 0.4082 29.394 2.98e-10 ***
The output in the ﬁrst column gives the estimated mean per group. The sec-
ond gives the standard error of each mean, the third the t-value (the estimate
divided by the standard error), and the last the corresponding p-values. From
the p-values the conclusion follows to reject the null hypotheses H
0
: µ
j
= 0
for Group index j running from 1 to 3.
Using the above design matrix, the model for the gene expression values
from diﬀerent groups can be written as
Y
ij
= µ
j
+ ε
ij
, where ε
ij
is distributed as N(0, σ
2
),
and Y
ij
is the expression of Person i in Group j, µ
j
the mean of Group j, and
the ε
ij
the error of Person i in Group j. The error is assumed to be normally
distributed with zero mean and variance equal for diﬀerent persons.
1
5.2. ONE-WAY ANALYSIS OF VARIANCE 77
The above illustrates that the linear model is useful for testing hypotheses
about group means. In bioinformatics the linear model is applied to many
sets of gene expressions, so that it is of great importance to have an overall
test for the equality of means.
5.2 One-way analysis of variance
A frequent problem is that of testing the null hypothesis that three or more
population means are equal. By comparing two types of variances, this is
made possible by a technique called analysis of variance (ANOVA). To set
the scene, let three groups of patients be available with measurements in the
form of gene expression values. The null-hypothesis to be tested is H
0
: µ
1
=
µ
2
= µ
3
. In statistical language such groups are called levels of a factor.
Let the data for Group 1 be represented by y
11
, y
21
, · · · , y
n1
those of Group
2 by y
12
, y
22
, · · · , y
n2
and those of Group 3 by y
13
, y
23
, · · · , y
n3
, where n is
the number of expression values in each group. The three sample means per
patient group can be expressed by
y
1
=
1
n
n

i=1
y
i1
, y
2
=
1
n
n

i=1
y
i2
, and y
3
=
1
n
n

i=1
y
i3
.
The total number of measurements N = 3n, so that the overall mean y is
equal to
y =
1
N
_
n

i=1
y
i1
+
n

i=1
y
i2
+
n

i=1
y
i3
_
.
For the deﬁnition of the overall test on the equality of means there are two
sums of squares of importance. The sum of squares within (SSW) is the sum
of the squared deviation of the measurements to their group mean, that is
SSW =
g

j=1
n

i=1
(y
ij
−y
j
)
2
,
where g is the number of groups. The sum of squares between (SSB) is the
sum of squares of the deviances of the group mean with respect to the total
mean, that is
SSB =
g

j=1
n

i=1
(y
j
−y)
2
= n
g

j=1
(y
j
−y)
2
.
78 CHAPTER 5. LINEAR MODELS
Now the f-value is deﬁned by
f =
SSB/(g −1)
SSW/(N −g)
.
If the data are normally distributed, then this f-value follows the F
g−1,N−g
distribution, where g −1 and N −g are the degrees of freedom (Rao, 1973,
p.245). If P(F
g−1,N−g
> f) ≥ α, then H
0
: µ
1
= µ
2
= µ
3
is not rejected, and,
otherwise it is. The idea behind the test is that, under the null-hypothesis
of equal group means, the value for SSB will tend to be small, so that the
observed f-value will be small and H
0
is accepted.
Example 1. Let’s continue with the data from the previous example.
Recall that the data of Group 1 are 2, 3, 1, 2, those of Group 2 are 8, 7, 9,
8, and of Group 3 are 11, 12, 13, 12. The number of expression values per
group n = 4, the total number of data values N = 12, and the number of
groups g = 3.
To load the data, to construct the corresponding factor, and to compute
the group means one may use the following.
> y <- c(2,3,1,2, 8,7,9,8, 11,12,13,12)
> a <- gl(3,4)
> gm <- as.numeric(tapply(y, a, mean))
> gm
[1] 2 8 12
Thus we ﬁnd that y
1
= 2, y
2
= 8, and y
3
= 12. These group means are
now collected in the vector gm. The grand mean y can be computed by
mean(y)=7.333333. An elementary manner to compute the sums of squares
between SSB is by
gm <- as.numeric(tapply(y, a, mean))
g <- 3; n <- 4; N <-12; ssb <- 0
for (j in 1:g) {ssb <- ssb + (gm[j]- mean(y))^2}
SSB <- n*ssb
This results in SSB = 202.6667. In a similar manner the sums of squares
within (SSW) and the f-value can be computed, as follows.
> SSW <- 0
> for (j in 1:g) {SSW <- SSW + sum((y[a==j]-gm[j])^2)}
> f <- (SSB/(g-1))/(SSW/(N-g))
5.2. ONE-WAY ANALYSIS OF VARIANCE 79
This results in SSW = 6 and an observed f-value equal to 152. Hence, the
overall p-value is
P(F
2,9
> 152) = 1 −P(F
2,9
< 152) = 1 −pf(152, 2, 9) = 1.159156 · 10
−7
.
Since this is smaller than the signiﬁcance level 0.05, the conclusion is to reject
the null hypothesis of equal means.
The built-in-function anova can be used to extract the so-called analysis
of variance table from an lm object.
> anova(lm(y ~ a))
Analysis of Variance Table
Response: x
Df Sum Sq Mean Sq F value Pr(>F)
fact 2 202.667 101.333 152 1.159e-07 ***
Residuals 9 6.000 0.667
This gives the degrees of freedom g − 1 = 2 and N − g = 9, the sums of
squares between (202.667), the sums of squares within (6.0), the f-value 152
and the overall p-value 1.159 · 10
−7
.
Example 2. By the previous analysis of variance it is concluded that
there are diﬀerences in population means. It is, however, not clear which of
the means diﬀer. A way to clarify this is by estimating the mean of Group 1
(Level 1) and then computing the diﬀerence between Group 2 and Group 1,
and the diﬀerence between Group 3 and Group 1. Such corresponds to the
following contrast matrix
C =
_
_
1 1 1
0 −1 0
0 0 −1
_
_
.
This contrast matrix is by default implemented by the model speciﬁcation
y~a, as follows.
> summary(lm(y ~ a))
Coefficients:
Estimate Std. Error t value Pr(>|t|)
(Intercept) 2.0000 0.4082 4.899 0.000849 ***
80 CHAPTER 5. LINEAR MODELS
factLevel 2 6.0000 0.5774 10.392 2.60e-06 ***
factLevel 3 10.0000 0.5774 17.321 3.22e-08 ***
Residual standard error: 0.8165 on 9 degrees of freedom
Multiple R-Squared: 0.9712, Adjusted R-squared: 0.9649
F-statistic: 152 on 2 and 9 DF, p-value: 1.159e-07
The estimated intercept is the mean of Group 1 (Level 1). The factLevel
2 is the diﬀerence in means between Group 2 (Level 2) and Group 1 and
factLevel 3 is the diﬀerence in means between Group 3 and Group 1. By
t-tests the null-hypothesis is tested that the mean of Group 1 is zero, the
diﬀerence in means between Group 2 and Group 1 is zero and the diﬀerence
in means between Group 3 and Group 1 is zero. That is, the null-hypotheses
are H
0
: µ
1
= 0, H
0
: µ
2
− µ
1
= 0, and H
0
: µ
3
− µ
2
= 0. Since the p-values
that correspond to the t-values are smaller than the signiﬁcance level 0.05, all
null-hypotheses are rejected. The last line of the output gives the f-value,
the degrees of freedom, and the corresponding overall p-value. The latter
equals that of ANOVA.
Before we analyze real gene expression data it seems well to give an ex-
ample where the means do not diﬀer.
Example 3. Let’s sample data from the normal distribution with mean
1.9 and standard deviation 0.5 corresponding to three groups of patients that
do not possess any type of diﬀerences between groups.
> y <- rnorm(12,1.9,0.5)
> round(x,2)
[1] 1.75 1.82 1.35 1.61 2.08 1.27 2.50 2.40 2.13 0.71 2.80 2.00
> a <- gl(3,4)
> anova(lm(y ~ a))$Pr[1] [1] 0.6154917 Note that by the$Pr[1] operator extracts the p-value from the list generated
by the anova function. The p-value implies the conclusion not to reject the
null-hypotheseis of equal means, which is consistent with the data generation
process.
5.2. ONE-WAY ANALYSIS OF VARIANCE 81
B1 B2 B3
3
.
5
4
.
0
4
.
5
5
.
0
5
.
5
Figure 5.1: Plot of SKI-like onco-
gene expressions for three patient
groups.
B1 B2 B3
6
.
0
6
.
2
6
.
4
6
.
6
6
.
8
7
.
0
7
.
2
Figure 5.2: Plot of Ets2 expression
values for three patient groups.
Example 4. B-cell ALL: 1866_g_at. To illustrate analysis of variance
by real data we shall use the ALL data from the ALL package, see Section
1.1. Speciﬁcally, expression levels from B-cell ALL patients in stage B1, B2,
and B3 are selected with row name 1866_g_at, which refers to an SKI-like
oncogene related to oncoproteins. From the plot of the data in Figure 5.1 it
can be observed that the expression levels diﬀer between the disease stages.
The null hypothesis is tested that the expression means in each stage are
equal or in other words that there are no experimental eﬀects. It is brieﬂy
indicated how the data are loaded.
> library(ALL);data(ALL)
> ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")] > y <- exprs(ALLB123)["1866_g_at",] > summary(lm(y ~ ALLB123$BT))
Estimate Std. Error t value Pr(>|t|)
(Intercept) 4.58222 0.08506 53.873 < 2e-16 ***
82 CHAPTER 5. LINEAR MODELS
ALLB123$BTB2 -0.43689 0.10513 -4.156 8.52e-05 *** ALLB123$BTB3 -0.72193 0.11494 -6.281 2.00e-08 ***
Residual standard error: 0.3707 on 75 degrees of freedom
Multiple R-squared: 0.3461, Adjusted R-squared: 0.3287
F-statistic: 19.85 on 2 and 75 DF, p-value: 1.207e-07
From the overall p-value 1.207 · 10
−7
of the f-test the conclusion follows to
reject the hypothesis of equal means. From the t-tests we conclude that the
mean of B1 diﬀers from zero and the diﬀerences between B2 and B1 as well
as between B3 and B2 are unequal to zero. That is, the population means
of Group B1, B2, and B3 do diﬀer.
Example 5. B-cell ALL: 1242_at. To illustrate a case where the means
do not diﬀer we selected the expression values for probe 1242_at of the B-
cell ALL patients in stage B1, B2, and B3 from the ALL data. This probe
corresponds to the Ets2 repressor factor which plays a role in telomerase
regulation in human cancer cells. From the plot of the data in Figure 5.2,
however, it can be observed that the expression values hardly diﬀer between
disease stages. The data are extracted from the ALL object and collected in
the vector y. The corresponding factor is given by ALLB123$BT. > library(ALL); data(ALL) > ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")]
> y <- exprs(ALLB123)["1242_at",]
> summary(lm(y ~ ALLB123$BT)) Estimate Std. Error t value Pr(>|t|) (Intercept) 6.55083 0.05673 115.483 <2e-16 *** ALLB123$BTB2 0.03331 0.07011 0.475 0.636
ALLB123$BTB3 -0.04675 0.07665 -0.610 0.544 Residual standard error: 0.2473 on 75 degrees of freedom Multiple R-squared: 0.01925, Adjusted R-squared: -0.006898 F-statistic: 0.7362 on 2 and 75 DF, p-value: 0.4823 From the overall p-value 0.4823, the conclusion is not to reject the null hy- pothesis of equal means. More speciﬁcally, the null-hypotheses H 0 : µ 1 = 0 is rejected, but from the p-value 0.636 the H 0 : µ 2 − µ 1 = 0 is not rejected, 5.3. TWO-WAY ANALYSIS OF VARIANCE 83 and from p-value 0.544 the H 0 : µ 3 −µ 2 = 0 is not rejected either. Example 6. An interesting question is of course for how many genes of the ALL data the hypothesis of equal means is rejected by the overall ANOVA p-value? Such can be answered by collecting the p-values in a vector. > pano <- apply(exprs(ALLB123),1,function(x) anova(lm(x~ALLB123$BT))$Pr[1]) > sum(pano<0.05) [1] 2526 Thus the hypothesis of equal means is rejected for 2526 out of a total of 12625 genes (probes). 5.3 Two-way analysis of variance Having some experience with one way analysis of variance, the question may arise whether the model for means of groups can be extended from one factor to more factors. This is indeed possible. The model would then be equal to Y ijk = α i + β j + (αβ) ij + ε ijk , where α i is the mean of Group i indicated by the ﬁrst factor, β j the mean of Group j indicated by the second factor, (αβ) ij the interaction eﬀect and ε ijk the error which is distributed according to N(0, σ 2 ). If the means of the i groups diﬀer, then there is a main eﬀect of the ﬁrst factor which is expressed in a p-value smaller than 0.05. Similarly, in case the means of the j groups diﬀer, there is a main eﬀect of the second factor, expressed in a p-value smaller than 0.05. Two-way analysis of variance will brieﬂy be illus- trated. Example 5. A two-way approach. It case of the ALL data from Chiaretty et al. (2004) we may aggregate the B cell patients into two groups: B, B1 and B2 in the ﬁrst and B3 and B4 in the second. For the second group we select from the molecular biology of the patients assigned to BCR/ABL and NEG. We shall perform the analysis on the expression values of NEDD4 binding protein 1 with probe id 32069_at. This can be computed as follows. library("ALL"); data(ALL) ALLBm <- ALL[,which(ALL$BT %in% c("B","B1","B2","B3","B4") & ALL$mol.biol %in% c("BCR/ABL","NEG"))] 84 CHAPTER 5. LINEAR MODELS facmolb <- factor(ALLBm$mol.biol)
facB <- factor(ceiling(as.integer(ALLBm$BT)/3),levels=1:2,labels=c("B012","B34")) > anova(lm(exprs(ALLBm)["32069_at",] ~ facB * facmolb)) Analysis of Variance Table Response: exprs(ALLBm)["32069_at", ] Df Sum Sq Mean Sq F value Pr(>F) facB 1 1.1659 1.1659 4.5999 0.0352127 * facmolb 1 3.2162 3.2162 12.6891 0.0006433 *** facB:facmolb 1 1.1809 1.1809 4.6592 0.0340869 * Residuals 75 19.0094 0.2535 First the patients are selected with B-cell ALL and assigned molecular biology of the cancer to BCR/ABL or NEG. Next the factors are constructed to group the patients. From the p-values in the analysis of variance table it can be concluded that there two main eﬀects as well as an interaction eﬀect. One may also ask for a summary of the individual eﬀects. > summary(lm(exprs(ALLBm)["32069_at",] ~ facB * facmolb)) Call: lm(formula = exprs(ALLBm)["32069_at", ] ~ facB * facmolb) Coefficients: Estimate Std. Error t value Pr(>|t|) (Intercept) 6.7649 0.1073 63.026 < 2e-16 *** facBB34 -0.5231 0.1686 -3.103 0.0027 ** facmolbNEG -0.6020 0.1458 -4.128 9.4e-05 *** facBB34:facmolbNEG 0.5016 0.2324 2.159 0.0341 * Residual standard error: 0.5034 on 75 degrees of freedom Multiple R-squared: 0.2264, Adjusted R-squared: 0.1954 F-statistic: 7.316 on 3 and 75 DF, p-value: 0.0002285 In bioinformatics the question often arises how many probes there are with have signiﬁcant eﬀects. In this case we may compute the number of probes with signiﬁcant main as well as interaction eﬀects. > pval <- apply(exprs(ALLBm), 1, function(x) anova(lm(x ~ facB * facmolb))$Pr[1:3])
5.4. CHECKING ASSUMPTIONS 85
> pvalt <- data.frame(t(pval))
> colnames(pvalt) <- c("maineffectB","maineffectmolbiol","interaction")
> sum(pvalt$maineffectB < 0.05 & pvalt$maineffectmolbiol < 0.05 & pvalt$interaction < 0.05) [1] 47 The three p-values per probe are collected in a matrix. This matrix is trans- posed so that the columns correspond to the p-values and the rows to the probes. Using the logical AND (&) operator and summing the TRUE values yield 47 probes with signiﬁcant main and interaction eﬀects. 5.4 Checking assumptions When the linear model is applied for analysis of variance there are in fact two assumptions made. First, the errors are assumed to be independent and normally distributed, and, second, the error variances are assumed to be equal for each level (patient group). The latter is generally known as the homoscedasticity assumption. The normality assumption can be tested as a null hypothesis by applying the Shapiro-Wilk test on the residuals. The homoscedasticity assumption can be tested as a hypothesis by the Breusch and Pagan (1979) test on the residuals. This latter test may very well be seen as a generalization of the F-test for equal variances. Example 1. Testing normality of the residuals. From Figure 5.1 it can be observed that there are outliers being far apart from the bulk of the other expression values. This raises the question whether the normality assumption holds. The normality of the residuals from the estimated linear model on the B-cell ALL data from 1866_g_at, can be tested as follows. > data(ALL,package="ALL");library(ALL) > ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")]
> y <- exprs(ALLB123)["1866_g_at",]
> shapiro.test(residuals(lm(y ~ ALLB123$BT))) Shapiro-Wilk normality test data: residuals(lm(y ~ ALLB123$BT))
W = 0.9346, p-value = 0.0005989
86 CHAPTER 5. LINEAR MODELS
From the p-value 0.0005989, the conclusion is to reject the null-hypothesis of
normally distributed residuals.
Example 2. Testing homoscedasticity of the residuals. From Figure
5.1 it can be observed that the spread of the expression values around their
mean diﬀers between groups of patients. In order to test the homoscedasticity
assumption we use the function bptest from the lmtest package.
> library(ALL); data(ALL); library(lmtest)
> ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")] > y <- exprs(ALLB123)["1866_g_at",] > bptest(lm(y ~ ALLB123$BT),studentize = FALSE)
Breusch-Pagan test
data: lm(y ~ ALLB123$BT) BP = 8.7311, df = 2, p-value = 0.01271 From the p-value 0.01271, the conclusion follows to reject the null hypothesis of equal variances (homoscedasticity). 5.5 Robust tests In case departures from normality or homoscedasticity are large enough to cause concern with respect to the actual signiﬁcance level or to the power of the test, an alternative testing procedure is called for. In case only ho- moscedasticity is violated, we are in a situation quite similar to that of t- testing with unequal variances. That is, the null hypothesis H 0 : µ 1 = µ 2 = µ 3 of equal means can be tested without assuming equal variances by a test proposed by Welch (1951). Example 1. In Example 2 of the previous section the hypothesis of equal variances was rejected. To apply analysis of variance without assuming equal variances (homoscedasticity) one may use the function oneway.test, as follows. > data(ALL,package="ALL");library(ALL) 5.5. ROBUST TESTS 87 > ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")]
> y <- exprs(ALLB123)["1866_g_at",]
> oneway.test(y ~ ALLB123$BT) One-way analysis of means (not assuming equal variances) data: y and ALLB123$BT
F = 14.1573, num df = 2.000, denom df = 36.998, p-value = 2.717e-05
From the p-value 2.717 · 10
−5
, the conclusion follows to reject the hypothesis
of equal means.
In case normality is violated a rank type of test is more appropriate. In
particular, to test the null-hypothesis of equal distributions of groups of gene
expression values, the Kruskal-Wallis rank sum test is recommended. This
test can very well be seen as a generalization of the Wilcoxon test for testing
the equality of two distributions. Because it is based on ranking the data,
it is highly robust against non-normality, it, however, does not estimate the
size of experimental eﬀects.
Example 2. In Example 1 of the previous section we rejected the hypoth-
esis of normally distributed residuals. We use the function kruskal.test to
perform a non-parametric test.
> data(ALL,package="ALL");library(ALL)
> ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")] > y <- exprs(ALLB123)["1866_g_at",] > kruskal.test(y ~ ALLB123$BT)
Kruskal-Wallis rank sum test
data: y by ALLB123$BT Kruskal-Wallis chi-squared = 30.6666, df = 2, p-value = 2.192e-07 From the p-value 2.192 · 10 −7 , the null-hypothesis of equal distributions of expression values between patient groups is rejected. By the apply functionality the p-values can easily be computed for all 12625 gene expression values of the ALL data. 88 CHAPTER 5. LINEAR MODELS 5.6 Overview and concluding remarks By applying the above normality and homogeneity tests to complete sets of gene expression values it can quickly be seen to what extent the assumptions for the classical analysis of variance test are violated. Based on these it can be decided to add rank type of testing in order to reduce the amount of false positives and false negatives. Here, false positives are signiﬁcant p-values for equal populations means and false negatives are non-signiﬁcant p-values for unequal populations means. In the next chapter it will brieﬂy be indicated how to combine two factors into a single analysis of variance. For instance, one may want to combine B-cell stage with age groups of persons. The interested reader is referred to Faraway (2004) and Venables & Ripley (2002) for more information on using linear models in R and for a general treatment of linear models to Rao & Toutenburg (1995). The p-values from overall tests of equality of means or distributions are important tools to order genes according to their experimental eﬀect with respect to diﬀerent patient groups. More examples are given in the next chapter where several functionalities of Bioconductor will be used for the analysis of microarray data. 5.7 Exercises 1. Analysis of gene expressions of B-cell ALL patients. (a) Construct a data frame containing the expression values for the B-cell ALL patients in stage B, B1, B2, B3, B4 from the ALL data. (b) How many patients are in each group. (c) Test the normality of the residuals from the linear model used for analysis of variance for all gene expression values. Collect the p-values in a vector. (d) Do the same for the homoscedasticity assumption. (e) How many gene expressions are normally distributed and how many homoscedastic? For how many do both hold? 2. Further analysis of gene expressions of B-cell ALL patients. Continue with the previous data frame containing the expression values for the 5.7. EXERCISES 89 B-cell ALL patients in stage B, B1, B2, B3, B4 from the ALL data. (a) Collect the overall p-values from ANOVA in a vector. (b) Use featureNames() to report the aﬀymetrix id’s of the genes with smaller p-values than 0.000001. (c) Collect the overall p-values from the Kruskal-Walles test in a vec- tor. (d) Use featureNames() to report the aﬀymetrix id’s of the genes with smaller p-values than 0.000001. (e) Brieﬂy comment on the diﬀerences you observe. That is, how many genes have p-values smaller than 0.001 from both ANOVA and Krusal-Wallis? How many only from one type of test? Hint: Collect TRUE/FALSES in logical vectors and use table. 3. Finding the ten best best genes among gene expressions of B-cell ALL patients. Continue with the previous data frame containing the expres- sion values for the B-cell ALL patients in stage B, B1, B2, B3, B4 from the ALL data. (a) Print the p-values and the corresponding (aﬃmetrix) gene identi- ﬁers of the ten best from ANOVA. (b) Do the same for the p-values from the Kruskal-Wallis test. (c) Use the function intersect to ﬁnd identiﬁers in both sets. 4. A simulation study on gene expression values. (a) Construct a data matrix with 10000 rows and 9 columns with data from the normal distribution with mean zero and variance equal to one. Such a matrix simulates gene expressions without diﬀerences between groups (sometimes called negatives). (b) Construct a factor for three groups each with three values. (c) How many p-values are smaller than the signiﬁcance level α = 0.05? (d) If the p-value is smaller than the signiﬁcance level, then the con- clusion is that there an experimental eﬀect (a positive). How many false positives do you expect and how many did you observe? 90 CHAPTER 5. LINEAR MODELS (e) Construct a matrix with 10000 rows and 9 columns with normally distributed data with mean zero, one and two and variance equal to one. Assume again that there three groups each with three data values. This data matrix simulates gene expressions with diﬀer- ences between groups (sometimes called positives). Use ANOVA and kruskal-Wallis to ﬁnd the number of signiﬁcant genes (true positives). report the number of true positives and false nega- tives. Chapter 6 Micro Array Analysis The analysis of gene expression values is of key importance in bioinformatics. The technique makes it possible to give an initial answer to many important genetic type of questions. In this chapter you learn how to preprocess probe data, ﬁlter genes, to program various visualizations, to use gene ontology identiﬁers, to load public available gene expression data, as well as how to summarize results in html output. 1 6.1 Probe data The microarray technique takes advantage of hybridization properties of nu- cleic acids. That is, to give a rough idea, complementary molecules are attached and labeled on a solid surface in order for a specialized scanner measure the intensity of target molecules. Per gene there are about twenty such measures obtained for each probe (gene). Per probe these measures come in pairs. The intensity of the perfect match (PM) intends to measure the amount of transcripts from the gene. The intensity of the mismatch (MM) is related to non-speciﬁc binding and is often seen as a background type of noise. The raw data from the Aﬀymetrix scanner is stored in so-called DAT ﬁles, which are processed to so-called CEL ﬁles, where we will work with. The package affy has facilities to read data from a vector specifying several CEL ﬁles produced by the Aﬀymetrix scanner. 1 It may be convenient to explore the possibilities of the limmaGUI. Our approach, however, will be to concentrate on the programming aspects using the commandline. 91 92 CHAPTER 6. MICRO ARRAY ANALYSIS Example 1. We will start with a built-in data set called MLL.B from the ALLMLL package. To load it and to retrieve basic information use > library(affy) > data(MLL.B, package = "ALLMLL") > MLL.B It is very useful to print the structure of the object str(MLL.B) and its slot names. > slotNames(MLL.B) [1] "cdfName" "nrow" "ncol" [4] "assayData" "phenoData" "featureData" [7] "experimentData" "annotation" ".__classVersion__" Additional information become available from str(MLL.B). The raw probe intensities are available from exprs(MLL.B), which extracts the probe in- tensities from the MLL.B object. The number of rows and columns of the expression values of MLL.B can be obtained by the dim function. > dim(exprs(MLL.B)) [1] 506944 20 The annotation can be extracted as follows. > annotation(MLL.B) [1] "hgu133b" To print the ﬁrst 10 names of the probes use > probeNames(MLL.B)[1:10] [1] "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" [6] "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" Note that the probe names are the same as those obtained by geneNames. The PM and MM values are collected by the functions pm and mm. To print the PM values of the ﬁrst four out of the sixteen rows of the probe with identiﬁer 200000_s_at we may use the following. 6.1. PROBE DATA 93 > pm(MLL.B,"200000_s_at")[1:4,1:3] JD-ALD009-v5-U133B.CEL JD-ALD051-v5-U133B.CEL JD-ALD052-v5-U133B.CEL 200000_s_at1 661.5 321.5 312.5 200000_s_at2 838.8 409.3 395.3 200000_s_at3 865.3 275.5 341.3 200000_s_at4 425.8 253.5 196.8 By function matplot a quick view on the variability of the data within and between probes can be obtained. > matplot(pm(MLL.B,"200000_s_at"),type="l", xlab="Probe No.", + ylab="PM Probe intensity") From the resulting plot in Figure 6.1 it can be observed that the variability is substantial. Density plots of the log of the probe values can be obtained by hist(MLL.B). From the density plot of the log of the intensity data in Figure 6.2 it can be seen that these are quite skew to the right. The script to program such plots 2 4 6 8 10 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 Probe No. P M P r o b e i n t e n s i t y Figure 6.1: Mat plot of intensity values for a probe of MLL.B. 6 8 10 12 14 0 . 0 0 . 2 0 . 4 0 . 6 0 . 8 1 . 0 1 . 2 log intensity d e n s i t y Figure 6.2: Density of MLL.B data. is quite brief. > MAplot(MLL.B,pairs=TRUE, plot.method= "smoothScatter") > image(MLL.B) 94 CHAPTER 6. MICRO ARRAY ANALYSIS 6.2 Preprocessing methods From various visualization methods it is clear that preprocessing of probe intensities is necessary for making biologically relevant conclusions. Biocon- ductor gives facilities for various preprocessing methods. Here we will only sketch what the main methods are and how these can be implemented. It should be noted that the topic of optimal preprocessing currently is a ﬁeld of intense research (probably for the coming years), so that deﬁnitive recom- mendations are not mandatory. Preprocessing consists of three major steps: Background correction, normalization, and summarization. To obtain the available background and pm correction methods use the following. > bgcorrect.methods [1] "mas" "none" "rma" "rma2" The mas background is part of the MAS Aﬀymetrix software and is based on the 2% lowest probe values. RMA uses only the PM values, neglects the MM values totally, and is based on conditional expectation and the normality assumption of probes values. There are also a number of correction methods available for the PM values: > pmcorrect.methods [1] "mas" "pmonly" "subtractmm" The following normalization methods are available: > normalize.methods(MLL.B) [1] "constant" "contrasts" "invariantset" "loess" [5] "qspline" "quantiles" "quantiles.robust" Constant is a scaling method equivalent to linear regression on a reference array although without intercept term. More general are the non-linear nor- malization methods such as loess, qspline, quantiles, and robust quantiles. Loess is a nonlinear method based on local regression of MA plots. The methods of contrasts is based on loess regression. Quantile normalization is an inverse transformation of the empirical distribution with respect to an averaged sample quantile in order to impose one and the same distribution to each array. The method qspline uses quantiles from each array and a target array to ﬁt a system of cubic splines. The target should be the mean (geo- metric) or median of each probe, but could also be the name of a particular group. 6.2. PREPROCESSING METHODS 95 The ﬁnal step of preprocessing is to aggregate multiple probe intensities into a gene expression value. The available methods are: > express.summary.stat.methods [1] "avgdiff" "liwong" "mas" "medianpolish" "playerout" The ﬁrst is the simplest as it is based on averaging. There is no single best method for all preprocessing problems. It seems, however, wise to use methods robust against outliers together with non-linear normalization methods. Example 1. The three pre-processing steps can be employed one after the other by the function expresso. To combine the background correction RMA with constant normalization and to use average diﬀerences for the computation of gene expression values, we may use the following. eset <- expresso(MLL.B,bgcorrect.method="rma", normalize.method="constant",pmcorrect.method="pmonly", summary.method="avgdiff") Example 2. Another frequently applied preprocessing method is RMA. It combines convolution background correction, quantile normalization, and summarization based on multi-array model ﬁt in a robust manner by a so- called median polish algorithm. > library(affy) > data(MLL.B, package = "ALLMLL") > eset3 <- rma(MLL.B) Background correcting Normalizing Calculating Expression > boxplot(data.frame(exprs(eset3))) The three stages of preprocessing by rma are part of the output. Before a box-and-whiskers plot can be constructed the expression values need to be extracted from the object eset3. After the foregoing it is often desirable to further preprocess the data in order to remove patient speciﬁc means or medians. When the patient me- dian is zero, for instance, testing for a gene to have mean expression value 96 CHAPTER 6. MICRO ARRAY ANALYSIS diﬀerent from zero becomes meaningful. Example 3. In the sequel we shall frequently work with the ALL data from the ALL package of Bioconductor. Here the data set is brieﬂy introduced (see also Section 1.1) and further processing steps are illustrated. The raw data have been jointly normalized by RMA and are available in the form of an exprSet object. 12625 gene expression values are available from microarrays of 128 diﬀerent persons suﬀering from acute lymphoblastic leukemia (ALL). A number of interesting phenotypical co-variates are available. For instance, the ALL$mol variable has TRUE/FALSE values for each of the 128 patients
depending on whether a reciprocal translocation occurred between the long
arms of Chromosome 9 and 22. This is casually related to chronic and acute
leukemia. One can also ask for table(ALL$BT) to obtain an overview of the numbers of patients which are in certain phases of a disease. See also the general help ?ALL for further information on the data or the article by Chiaretti et al. (2004). > data(ALL, package = "ALL") > slotNames(ALL) [1] "assayData" "phenoData" "featureData" [4] "experimentData" "annotation" ".__classVersion__" > row.names(exprs(ALL))[1:10] [1] "1000_at" "1001_at" "1002_f_at" "1003_s_at" "1004_at" "1005_at" [7] "1006_at" "1007_s_at" "1008_f_at" "1009_at" By feno <- pData(ALL) phenotypical information from the patients is stored in a data frame, which is useful for further analysis. In case the gene expres- sion values over the patients are non-normally distributed one may want to subtract the median and divide by the MAD. An eﬃcient manner to do so is to use an apply function to compute the column mad and median, and sweep to subtract the median from each column entry and, next, to divide each column entry by the MAD. ALL1pp <- ALL1 <- ALL[,ALL$mol == "ALL1/AF4"]
meds <- apply(exprs(ALL1), 2, median)
dat <- sweep(exprs(ALL1), 2, meds)
exprs(ALL1pp) <- sweep(dat, 2, mads, FUN="/")
6.3. GENE FILTERING 97
By this script the patients are selected with assigned molecular biology
equal to ALL1/AF4. Then ALL1 is copied in order to overwrite the expression
values in a later stage. The median and the MAD are computed per column
by the speciﬁcation 2 (column index) in the apply function. Then the ﬁrst
sweep function subtracts the medians from the expression values and second
divides these by the corresponding MAD. By comparing the box plots in
Figure 6.3 and 6.4 the eﬀect of preprocessing can be observed. The medians
of the preprocessed data are equal to zero and the variation is smaller due
to the division by their MAD. Note that by box plotting a data frame a fast
overview of the distributions of columns in a data frame is obtained.
X04006 X16004 X24005 X28028 X31007
2
4
6
8
1
0
1
2
1
4
Figure 6.3: Boxplot of the
ALL1/AF4 patients.
X04006 X16004 X24005 X28028 X31007

1
0
1
2
3
4
Figure 6.4: Boxplot of the
ALL1/AF4 patients after median
6.3 Gene ﬁltering
A few important methods to ﬁlter genes are illustrated here. It is wise to
keep in mind that there are statistical as well as and biological criteria for
ﬁltering genes and that a combination of these often gives the most satisfac-
tory results. The examples stress the importance of careful thinking.
98 CHAPTER 6. MICRO ARRAY ANALYSIS
Example 1. Filtering by the coeﬃcient of variation. A manner to ﬁlter
genes is by the coeﬃcient of variation, which is deﬁned as the standard
deviation divided by the absolute value of the mean: cv = σ/|µ|. If cv = 1,
then the standard deviation equals the mean, so that the experimental eﬀect
is small relative to the precision of measurement. If, however, cv < 0.2, then
the mean is ﬁve times larger than the standard deviation, so that both the
experimental eﬀect and the measurement precision are large. Let’s compute
the coeﬃcient of variation per gene for the ALL1pp data of the previous
section.
> cvval <- apply(exprs(ALL1pp),1,function(x){sd(x)/abs(mean(x))})
Now using sum(cvval<0.2) yields 4751 genes with a coeﬃcient of variation
smaller than 0.2. These genes can be selected by ALL1pp[cvval<0.2,].
Example 2. Combining several ﬁlters. It is often desired to combine
several ﬁlters. Of course it is possible to program ﬁlters completely on your
own, however, we may conveniently use the function filterfun to combine
several ﬁlters. The script in this example is useful when several functions are
to be applied to a single data set.
library("genefilter")
f1 <- function(x)(IQR(x)>0.5)
f2 <- pOverA(.25, log2(100))
f3 <- function(x) (median(2^x) > 300)
f4 <- function(x) (shapiro.test(x)$p.value > 0.05) f5 <- function(x) (sd(x)/abs(mean(x))<0.1) f6 <- function(x) (sqrt(10)* abs(mean(x))/sd(x) > qt(0.975,9)) ff <- filterfun(f1,f2,f3,f4,f5,f6) library("ALL"); data(ALL) selected <- genefilter(exprs(All[,ALL$BT=="B"]), ff)
After running this script and using sum(selected) one obtains 317 genes
that pass the combined ﬁlter. The ﬁrst function returns TRUE if the in-
terquartile range is larger than 0.5, the second if 25% of the gene expression
values is larger than 6.643856, the third if the median of the expression values
taken as powers to the base two is larger than 300, the fourth if it passes the
Shapiro-Wilk normality test, the ﬁfth if the coeﬃcient of variation is smaller
than 0.1, and the sixth if the one-sample t-value is signiﬁcant. The ﬁlter
6.3. GENE FILTERING 99
functions are combined by filterfun and the function genefilter returns
a logical vector indicating whether the gene passed all the ﬁlters or failed
at least one of them. In order to use these ﬁlter steps properly it is well to
think them through because several ﬁlters focus on similar properties. In
particular, since the IQR divided by 1.349 is a robust estimator of the stan-
dard deviation, the ﬁrst ﬁlter selects genes with a certain minimal standard
deviation. With respect to the third ﬁlter note that 2
x
> 300 is equivalent
to x >
2
log(300) ≈ 8.228819, which is highly similar to the second ﬁlter.
Furthermore, s/|x| < 0.1 is equivalent to

10|x|/s > 1/

10, so that the last
two ﬁlters are highly similar.
Example 3. Filtering by t-test and normality. One may also want to
select genes with respect to p-values of a two-sample t-test over B-cell ALL
versus T-cell ALL. This can be combined with a normality test in the sense
that only those genes are ﬁltered which pass the Shapiro-Wilk normality test.
The latter will be applied separately for the B-cell ALL patients and for the
T-cell ALL patients. For this we write a function that will be used twice.
First, however, we create a logical factor patientB indicating patients with
B-cell ALL (TRUE) and with T-cell ALL (FALSE). The ﬁlter deﬁned selects
genes that have their p-value from the Welch two-sample t-test smaller than
the signiﬁcance level 0.05. A logical variable named selected is deﬁned
which attains TRUE only if sel1, sel2, as well as sel3 have the value
TRUE.
library("genefilter");library("ALL"); data(ALL)
patientB <- factor(ALL$BT %in% c("B","B1","B2","B3","B4")) f1 <- function(x) (shapiro.test(x)$p.value > 0.05)
f2 <- function(x) (t.test(x ~ patientB)$p.value < 0.05) sel1 <- genefilter(exprs(ALL[,patientB==TRUE]), filterfun(f1)) sel2 <- genefilter(exprs(ALL[,patientB==FALSE]), filterfun(f1)) sel3 <- genefilter(exprs(ALL), filterfun(f2)) selected <- sel1 & sel2 & sel3 ALLs <- ALL[selected,] This gives 1817 genes which pass the three ﬁlters. For these genes it holds that the expression values for B-cell ALL patients as well as for T-cell ALL patients are normally distributed (in the sense of non-rejection). A fundamental manner to visualize how the genes are divided among ﬁlters is 100 CHAPTER 6. MICRO ARRAY ANALYSIS by construction of a Venn diagram. This can conveniently be done by using functions from the limma package (Smyth, 2005). library(limma) x <- matrix(as.integer(c(sel1,sel2,sel3)),ncol = 3,byrow=FALSE) colnames(x) <- c("sel1","sel2","sel3") vc <- vennCounts(x, include="both") vennDiagram(vc) From the resulting Venn diagram in Figure 6.5 it can be seen that 1817 genes pass all three ﬁlters, 1780 genes pass none, 3406 genes pass the normality tests but not the t-test ﬁlter, etc. sel1 sel2 sel3 1780 920 2151 1366 826 359 3406 1817 Figure 6.5: Venn diagram of se- leced ALL genes. X04006 X16004 X24005 X28028 X31007 1 0 1 2 3 4 Figure 6.6: Boxplot of the ALL1/AF4 patients after median subtraction and MAD division. 6.4 Applications of linear models The limma package is frequently used for analyzing microarray data by linear models, such as ANOVA. 6.4. APPLICATIONS OF LINEAR MODELS 101 Example 1. Analysis of variance. We select patients with B-cell leukemia in a beginning stage B and in more progressive stages B1 and B2. The type of analysis is speciﬁed by using a factor that deﬁnes the model (design) matrix. Then the linear model is ﬁtted to the data and an empirical Bayes procedure is used to adapt the gene speciﬁc variances with a global variance estimator (Smyth, 2004) 2 . library("ALL"); library("limma"); data(ALL, package = "ALL") allB <- ALL[,which(ALL$BT %in% c("B","B1","B2"))]
design.ma <- model.matrix(~ 0 + factor(allB$BT)) colnames(design.ma) <- c("B","B1","B2") fit <- lmFit(allB, design.ma) fit <- eBayes(fit) > toptab <- topTable(fit, coef=2,5,adjust.method="fdr") > print(toptab[,1:5],digits=4) ID logFC AveExpr t P.Value 12586 AFFX-hum_alu_at 13.42 13.50 326.0 3.165e-99 2488 32466_at 12.68 12.70 306.3 1.333e-97 2773 32748_at 12.08 12.11 296.3 9.771e-97 5328 35278_at 12.44 12.45 295.5 1.146e-96 4636 34593_g_at 12.64 12.58 278.0 4.431e-95 By topTable the ﬁve genes are selected with the smallest p-values adjusted for the false discovery rate. Let’s call the mean of the B patients µ, that of B1 µ 1 , and that of B2 µ 2 . In the current case we are not so much interested in the hypothesis H 0 : µ − µ 2 , because this is the diﬀerence between Stage 0 and Stage 3. Rather, we are interested in the hypothesis H 0 : µ − µ 1 and H 0 : µ 1 − µ 2 . Such a speciﬁc hypothesis can be tested by using a contrast matrix, which can be speciﬁed as follows. > cont.ma <- makeContrasts(B-B1,B1-B2, levels=factor(allB$BT))
> cont.ma
Contrasts
Levels B - B1 B1 - B2
B 1 0
B1 -1 1
B2 0 -1
2
To obtain the appropriate number of levels we make a factor of ALLB$BT. 102 CHAPTER 6. MICRO ARRAY ANALYSIS Observe that the contrast matrix speciﬁes the diﬀerence between the levels B and B1 as well as between B1 and B2. It can be implemented as follows. fit1 <- contrasts.fit(fit, cont.ma) fit1 <- eBayes(fit1) toptabcon <- topTable(fit, coef=2,5,adjust.method="fdr") print(toptabcon[,1:5],digits=4) > toptabcon <- topTable(fit1, coef=2,5,adjust.method="fdr") > print(toptabcon[,1:5],digits=4) ID logFC AveExpr t P.Value 3389 33358_at 1.4890 5.260 7.374 5.737e-10 419 1389_at -1.7852 9.262 -7.081 1.816e-09 1016 1914_at 2.0976 4.939 7.019 2.315e-09 6939 36873_at 1.8646 4.303 6.426 2.361e-08 7542 37471_at 0.8701 6.551 6.106 8.161e-08 Here, we have applied a method called “false discovery rate” (fdr) which in- creases the p-values somewhat in order to reduce the number false positives. The number of genes requested equals 5. A very convenient manner to summarize, collect, and communicate vari- ous types of results is in the form of an HTML ﬁle. Example 2. Summarizing output in HTML format. It is often desired to combine the typical output from a function like topTable with that of an HTML output page containing various types of information. To illustrate this we proceed with the object toptabcon of the previous example. library("annaffy");library("hgu95av2.db") anntable <- aafTableAnn(as.character(toptabcon$ID), "hgu95av2.db", aaf.handler())
saveHTML(anntable, "ALLB123.html", title = "B-cell 012 ALL")
By the function aafTableAnn various types of information are gathered from
the output topTable of the estimated linear model, the annotation package,
and the aaf.handler functionality. The information collected contains the
following: Probe, Symbol, Description, Function, Chromosome, Chromo-
some Location, GenBank, LocusLink, Cytoband, UniGene, PubMed, Gene
Ontology, and Pathway. The resulting anntable is saved in HTML format
in the working directory or the Desktop. It contains a wealth of information
6.4. APPLICATIONS OF LINEAR MODELS 103
on e.g. Chromosome location, KEGG mappings, summaries from Pubmed
articles, etc.
Example 3. Using basic R functions. It is also possible to summarize
results in an HTML table on the basis of p-values from e.g. analysis of
variance (ANOVA). That is, the selected genes can directly be used as input
for aafTableAnn.
library("multtest"); library("annaffy"); library("hgu95av2.db")
library("ALL"); data(ALL, package = "ALL")
ALLB <- ALL[,which(ALL$BT %in% c("B","B1","B2"))] panova <- apply(exprs(ALLB), 1, function(x) anova(lm(x ~ ALLB$BT))$Pr[1]) genenames <- featureNames(ALLB)[panova<0.000001] atab <- aafTableAnn(genenames, "hgu95av2.db", aaf.handler()[c(1:3,8:9,11:13)]) saveHTML(atab, file="ANOVAonB-cellGroups.html") hgu95av2.db is a meta data annotation package connecting the requested information by the call to aaf.handler. The meaning of the columns can be obtained from the help page of the function. The resulting table is saved as an HTML ﬁle in the working directory (getwd()) or desktop. In a similar manner the p-values from the Kruskal-Wallis test can be used to select genes. Bioconductor has a useful facility to download publicly available microar- ray data sets from NCBI. Example 4. Analyzing public available data. The GDS1365 data con- tain primed macrophage response to IFN-gamma restimulation after diﬀerent time periods. The purpose is to gain insight into the inﬂuence of IFN-gamma priming on IFN-gamma induced transcriptional responses. Among the phe- notypical covariates of the data there is a factor time with levels 0, 3 and 24 hours and a factor protocol with the levels ”IFN-gamma primed” and ”un- primed”, which can be extracted by the function pData. Since researchers are often interested in the interaction between factors, we shall select genes with a signiﬁcant interaction eﬀect. library(GEOquery); library(limma); library(hgu95av2.db);library(annaffy) gds <- getGEO("GDS1365") eset <- GDS2eSet(gds,do.log2=T) 104 CHAPTER 6. MICRO ARRAY ANALYSIS prot <- pData(eset)$protocol
time <- pData(eset)$time pval <- apply(exprs(eset)[1:12625,], 1, function(x) anova(lm(x ~ prot * time))$Pr[1:3])
pvalt <- data.frame(t(pval))
colnames(pvalt) <- c("meffprot","mefftime","interaction")
genenames <- featureNames(eset)[pvalt$meffprot< 0.01 & pvalt$mefftime < 0.01 & pvalt$interaction < 0.01] atab <- aafTableAnn(genenames,"hgu95av2.db",aaf.handler()[c(1:3,8:9,11:13)]) saveHTML(atab, file="Two-way ANOVA protocol by time.html") By getGEO the data are downloaded to the disk and next these can be loaded into the R system. By GDS2eSet these are transformed to an expression set so that these can be analyzed statistically. The function pData extracts the factors from the expression set eset. The function anova extracts the p- value of the interaction eﬀect from the estimated linear model. We restrict the analysis to the ﬁrst 12625 rows because the additional ones contain not available values. The resulting html ﬁle seems to contain may interesting genes. 6.5 Searching an annotation package Detailed information on microarray experiments is stored in an annotation package. > library("ALL"); data(ALL) > annotation(ALL) [1] "hgu95av2" Hence, the annotation package we need is hgu95av2.db. Let’s load it and obtain an overview of its functionality. > library(hgu95av2.db) > ls("package:hgu95av2.db") [1] "hgu95av2" "hgu95av2_dbconn" "hgu95av2_dbfile" [4] "hgu95av2_dbInfo" "hgu95av2_dbschema" "hgu95av2ACCNUM" [7] "hgu95av2ALIAS2PROBE" "hgu95av2CHR" "hgu95av2CHRLENGTHS" [10] "hgu95av2CHRLOC" "hgu95av2CHRLOCEND" "hgu95av2ENSEMBL" 6.5. SEARCHING AN ANNOTATION PACKAGE 105 [13] "hgu95av2ENSEMBL2PROBE" "hgu95av2ENTREZID" "hgu95av2ENZYME" [16] "hgu95av2ENZYME2PROBE" "hgu95av2GENENAME" "hgu95av2GO" [19] "hgu95av2GO2ALLPROBES" "hgu95av2GO2PROBE" "hgu95av2MAP" [22] "hgu95av2MAPCOUNTS" "hgu95av2OMIM" "hgu95av2ORGANISM" [25] "hgu95av2PATH" "hgu95av2PATH2PROBE" "hgu95av2PFAM" [28] "hgu95av2PMID" "hgu95av2PMID2PROBE" "hgu95av2PROSITE" [31] "hgu95av2REFSEQ" "hgu95av2SYMBOL" "hgu95av2UNIGENE" [34] "hgu95av2UNIPROT" The annotation package contains environments with diﬀerent types of infor- mation. An easy manner to make the content of an environment available is by converting it into a list and to print part of it to the screen. > ChrNrOfProbe <- as.list(hgu95av2CHR) > ChrNrOfProbe[1]$‘1000_at‘
[1] "16"
We recognize the manufacturers identiﬁer of genes and the corresponding
chromosome. Asking information by ?hgu95av2CHR reveals that it is an
environment (hash table) which provides mappings between identiﬁers and
chromosomes. From these we obtain various types of information on the
basis of the manufacturer’s identiﬁer such as "1389_at". Below we obtain,
respectively, the GenBank accession number, the Entrez Gene identiﬁer, the
gene abbreviation, gene name, brief summaries of functions of the gene prod-
ucts, and the UniGene identiﬁer. For this we use the get function in order
to search an environment for a name.
> get("1389_at", env = hgu95av2ACCNUM)
[1] "J03779"
> get("1389_at", env = hgu95av2ENTREZID)
[1] 4311
> get("1389_at", env = hgu95av2SYMBOL)
[1] "MME"
> get("1389_at", env = hgu95av2GENENAME)
[1] "membrane metallo-endopeptidase (neutral endopeptidase,
enkephalinase, CALLA, CD10)"
> get("1389_at", env = hgu95av2SUMFUNC)
[1] NA
106 CHAPTER 6. MICRO ARRAY ANALYSIS
> get("1389_at", env = hgu95av2UNIGENE)
[1] "Hs.307734"
Let’s use the GenBank accession number to search its nucleotide data base.
> library(annotate)
> genbank("J03779",disp="browser")
From this we obtain the corresponding GI:179833 number, which can be used
to obtain a complete XML document.
> genbank(1430782,disp="data",type="uid")
Obviously, probes correspond to genes and frequently we are interested in
their chromosome location, and, speciﬁcally, in starting position(s).
> get("1389_at", env = hgu95av2CHRLOC)
3 3 3
156280152 156280327 156280748
Its cytoband location can also be obtained.
> get("1389_at", env = hgu95av2MAP)
[1] "3q25.1-q25.2"
Hence, we see that the gene is on Chromosome 3 at q arm band 25 sub-
band 1 and 2. In case we have a LocusLink ID, e.g. 4121, available the
corresponding GO terms can be obtained and stored in a list.
ll1<-GOENTREZID2GO[["4121"]]
6.6 Using annotation to search literature
Given the manufactures probe identiﬁer it is possible to search literature by
collecting Pubmed ID’s and to use these to collect relevant articles.
> library(hgu95av2.db);library(annotate); library(ALL); data(ALL)
> pmid <- get("1389_at",env=hgu95av2PMID)
> pubmed(pmid,disp="browser")
Another possibility is to collect a list containing PubMed ID, authors, ab-
stract text, title, journal, and publication date.
6.7. SEARCHING GO NUMBERS AND EVIDENCE 107
> absts <- pm.getabst("1389_at", "hgu95av2")
> pm.titles(absts)
The list can obviously be searched for regular expressions.
ne <- pm.abstGrep("neutral endopeptidase",absts[[1]])
Another possibility is to construct an HTML table with the titles.
> pmAbst2HTML(absts[[1]],filename="pmon1389_at.html")
6.7 Searching GO numbers and evidence
By the phrase “ontology” we mean a structured language about some con-
ceptual domain. The gene ontology consortium deﬁnes three ontologies: A
Molecular Function (MF) describes a phenomenon at the biochemical level
such as “enzyme”, “transporter”, or “ligand”. A Biological Process (BP)
may coordinate various related molecular functions such as “DNA replica-
tion” or “signal transduction”. A Cellular Component (CC) is a unit within
a part of the cell such as “chromosome”, “nucleus”, or “ribosome”.
Each term is identiﬁed by a unique GO number. To ﬁnd GO numbers
and their dependencies we use get to extract a list from the annotation ﬁles
hgu95av2GO for example. From the latter we extract a list and use an apply
type of function to extract another list containing GO identiﬁcation numbers.
> go1389 <- get("1389_at", env = hgu95av2GO)
> idl <- lapply(go1389,function(x) x$GOID) > idl[[1]] [1] "GO:0006508" The list idl contains 8 members of which only the ﬁrst is printed to the screen. By changing GOID into Ontology more speciﬁc information pertaining to ontology is extracted. From the annotate package we may now select the GO numbers which are related to a biological process. > library(annotate) > getOntology(go1389,"BP") [1] "GO:0006508" "GO:0007267" 108 CHAPTER 6. MICRO ARRAY ANALYSIS There are various types of evidence such as: inferred from genetic interaction (IGI), inferred from electronic annotation (IEA), traceable author statement (TAS), etc. Per GO identiﬁer the type of evidence can be obtained. > getEvidence(go1389) GO:0004245 GO:0005886 GO:0005887 GO:0006508 GO:0007267 GO:0008237 GO:0008270 "IEA" "TAS" "TAS" "TAS" "TAS" "TAS" "IEA" GO:0046872 "IEA" When we now want to select the GO numbers with evidence of a traceable author statement we can use the subset function to create a list. go1389TAS <- subset(go1389,getEvidence(go1389)=="TAS") A manner to extract information from this list is by using an apply type of function. > sapply(go1389TAS,function(x) x$GOID)
> sapply(go1389TAS,function(x) x$Evidence) > sapply(go1389TAS,function(x) x$Ontology)
We shall use this list in the below.
6.8 GO parents and children
The term “transmembrane receptor protein-tyrosine kinase” is more speciﬁc
and therefore a ’child’ of the more general term parent term “transmembrane
receptor” (Gentleman, et. al, 2005).
Example 1. Collecting GO information. There are functions to obtain
parents and children from a GO identiﬁer.
> GOMFPARENTS$"GO:0003700" isa isa "GO:0003677" "GO:0030528" > GOMFCHILDREN$"GO:0003700"
isa
"GO:0003705"
6.9. GENE FILTERING BY A BIOLOGICAL TERM 109
In case of a list of GO identiﬁers you may want to collect the ontology,
parents, and children identiﬁers in a vector.
go1389 <- get("1389_at", env = hgu95av2GO)
gonr <- getOntology(go1389, "BP")
gP <- getGOParents(gonr)
gC <- getGOChildren(gonr)
gPC <- c(gonr,gP,gC)
pa <- sapply(gP,function(x) x$Parents) ch <- sapply(gC,function(x) x$Children)
gonrc <- c(gonr,unlist(pa),unlist(ch))
Example 2. Probe selection by GO. A research strategy may be to start
with a probe number, to ﬁnd the GO identiﬁers of the biological process, to
obtains its parents, and next to transform these to probes.
library(GO); library(annotate); library("ALL"); data(ALL)
go1389 <- get("1389_at", env = hgu95av2GO)
gonr <- getOntology(go1389, "BP")
gP <- getGOParents(gonr)
pa <- sapply(gP,function(x) x$Parents) probes <- mget(pa,hgu95av2GO2ALLPROBES) probeNames <- unlist(probes) ALLpr <- ALL[probeNames,] > dim(exprs(ALLpr)) [1] 7745 128 Indeed, you may end up with many genes, useful for further analysis. 6.9 Gene ﬁltering by a biological term An application of working with GO numbers is to ﬁlter for genes which are related to a biological term. Example 1. Filter gene by a term. From a biological point of view it is most interesting to select genes which are related to a certain biolog- ical process to be speciﬁed by a term such as ”transcriptional repression”. 110 CHAPTER 6. MICRO ARRAY ANALYSIS We combine this with the previous ﬁlter. For this we need the annota- tion package used in the stage of data collection. This can be obtained by annotation(ALL). First we deﬁne a function (Gentleman, et al., 2005, p. 123) to collect appropriate GO numbers from the environment GOTERM. library("GO"); library("annotate"); library("hgu95av2.db") GOTerm2Tag <- function(term) { GTL <- eapply(GOTERM, function(x) {grep(term, x@Term, value=TRUE)}) Gl <- sapply(GTL, length) names(GTL[Gl>0]) } > GOTerm2Tag("transcriptional repressor") [1] "GO:0016564" "GO:0016565" "GO:0016566" "GO:0017053" The functions eapply and sapply search an environment like GOTERM by grep for matches of a speciﬁed term. A precaution is taken to select only those names which are not empty. This gives the GO terms which can now be translated to probe of the ALLs data. tran1 <- hgu95av2GO2ALLPROBES$"GO:0016564"
tran2 <- hgu95av2GO2ALLPROBES$"GO:0016566" tran3 <- hgu95av2GO2ALLPROBES$"GO:0017053"
tran <- c(tran1,tran2,tran3)
inboth <- tran %in% row.names(exprs(ALLs))
ALLtran <- ALLs[tran[inboth],]
The GO translated probe names are intersected with the row names of the
data giving the logical variable inboth. The variable tran[inboth] gives
the ids by which genes can be selected. Next, gene ids for which inboth
equals TRUE are selected and the corresponding data are collected in the data
frame ALLtran. More information can be obtained by GOTERM$"GO:0016564. By dim(exprs(ALLtran)) it can be observed that 26 genes which passed the normality ﬁlter are related to ”transcriptional repression”. 6.10 Signiﬁcance per chromosome After a statistical analysis to ﬁlter and order genes it is often quite useful to do post analysis on the results. In particular, after collecting p-values from 6.10. SIGNIFICANCE PER CHROMOSOME 111 a t-test one may wonder whether genes with signiﬁcant p-values occur more often within a certain chromosome. To test for such over or under represen- tation the Fisher test is very useful (see Section 4.1.7). Example 1. On the expression values of the ALL data we perform a two sample t-test using the patient group for which remission was achieved and for which it was not achieved. Per chromosome it can be tested whether the odds ratio diﬀers from 1 or, equivalently, whether there is independence. The data for the test consist of the number of signiﬁcant probes on Chromosome 19, the number of non-signiﬁcant probes on Chromosome 19, the number of remaining signiﬁcant probes, and the number of remaining non-signiﬁcant probes. > library("ALL"); data(ALL); library("hgu95av2.db") > rawp <- apply(exprs(ALL), 1, function(x) t.test(x ~ ALL$remission)$p.value) > xx <- as.list(hgu95av2CHR) > AffimIDChr <- names(xx[xx=="19"]) > names(rawp) <- featureNames(ALL) > f <- matrix(NA,2,2) > f[1,1] <- sum(rawp[AffimIDChr]<0.05); f[1,2] <- sum(rawp[AffimIDChr]>0.05) > f[2,1] <- sum(rawp<0.05) - f[1,1] ; f[2,2] <- sum(rawp>0.05) - f[1,2] > print(f) [,1] [,2] [1,] 106 638 [2,] 832 11049 > fisher.test(f) Fisher’s Exact Test for Count Data data: f p-value = 4.332e-11 alternative hypothesis: true odds ratio is not equal to 1 95 percent confidence interval: 1.757949 2.748559 sample estimates: odds ratio 2.206211 112 CHAPTER 6. MICRO ARRAY ANALYSIS > chisq.test(f) Pearson’s Chi-squared test with Yates’ continuity correction data: f X-squared = 52.3803, df = 1, p-value = 4.573e-13 The number of signiﬁcant probes is larger for Chromosome 19 resulting in an odds ratio of 2.2. The hypothesis of independence is rejected by both tests. 6.11 Overview and concluding remarks Many examples are given on using analysis of variance or T-tests for select- ing genes with large experimental eﬀects on diﬀerent patient groups. The above statistical methods seem to cover the majority of problems occurring in practice. 6.12 Exercises 1. Gene ﬁltering on normality per group of B-cell ALL patients. (a) Use genefilter to program the Shapiro normality test separately for each gene of the groups ”B1”,”B2”,”B3”,”B4”. (b) How many pass the ﬁlter? (c) Compute a Venn diagram for group ”B2”, ”B3”, and ”B4”, plot it, and give a correct interpretation for each number. 2. Analysis of gene expressions of B-cell ALL patients using Limma. (a) Construct a data frame containing the expression values for the B-cell ALL patients in stage B, B1, B2, B3, B4 from the ALL data. (b) Construct the design matrix and an appropriate contrast matrix. (c) Compute the twenty best genes by topTable. (d) Collect information on the twenty best gene s in an HTML page. 6.12. EXERCISES 113 3. Finding a row number. Use grep to ﬁnd the row number of gene 1389_at. Hint: Use row.names or featureNames. 4. Remission (genezing) from acute lymphocytic leukemia (ALL). With respect to the ALL data from the ALL library there is a phenotypical vari- able called remission indicating complete remission CR or refractory REF meaning improvement from disease and less or no improvement, respectively. (a) How many persons are classiﬁed as CR and REF, respectively? Hint: Use pData to extract a data frame with phenotypical data. (b) Program the two-sample t-test not assuming equal variances to select genes with p-values smaller than 0.001. Hint: You may have to select the persons with values on remission, excluding not available data. (c) Collect and give the manufactures probe names of the genes with p-values smaller than 0.001. (d) Use the probe names to ﬁnd the corresponding gene names. Give the code. (e) Is the famous protein p53 is among them? (f) How many unique gene names are there? 5. Remission achieved. For the ALL data from its ALL library the patients are checked for achieving remission. The variable ALL$CR has values CR
(became healthy) and REF (did not respond to therapy; remain ill).
(a) Construct a separate data frame consisting of only those gene
expression values from patients that have values CR or REF.
(b) How many genes have a p-value smaller than 0.0001 from the two-
sample T-test not assuming equal variances? Hint: Use the apply
functionality to program the test.
(c) Give the aﬀymetrix names (symbols) of the genes the pass the
selection criterion of p-value smaller than 0.0001.
(d) Use the latter to ﬁnd the biological names.
(e) How many oncogenes are there is total?
114 CHAPTER 6. MICRO ARRAY ANALYSIS
(f) Do the Fisher test on the number of oncogenes out of the total
versus the number of signiﬁcant oncogenes out of the selected.
6. Gene ﬁltering of ALL data. The data are in the library called ”ALL”.
The persons with T-cell leukemia which are in stage T2 and T3 can
be selected by the variable ALL$BT. You may use the function ”table” to ﬁnd the frequencies of the patient types and leukemia stages. To answer the questions below functions from the library ”geneﬁlter” are helpful. (a) Program a gene ﬁlter step separately for T2 and T3 patients such that only those genes pass which are normally distributed. (b) Program a second ﬁlter step which passes only those genes with a signiﬁcant p-value from the two sample T-test. (c) How many genes pass all ﬁlter steps? (d) How many genes pass normality? 7. Stages of B-cell ALL in the ALL data. Use the limma package to answer the questions below. (a) Select the persons with T-cell leukemia which are in stage B1, B2, B3, and B4. (b) What type of contrast matrix would you like to suggest in this situation? Give its code. (c) Perform analysis of variance to test the hypothesis of equal pop- ulation means. Use the Benjamini & Hochberg (1995) (”BH”) adjustment method for the false discovery rate and topTable to report the ﬁve best genes. (d) For how many genes is the null-hypothesis to be rejected? 8. Analysis of public micro array data on rheumatoid arthritis. (a) Download GDS486 and transform it into eset form. Here we meet a missing data problem. A manner to solve it is as follows. Use the function function(x) sum(is.na(x)) in apply on the rows to count the number of missing values per row. Select the rows without missing value to perform a two-sample t-test with the 6.12. EXERCISES 115 groups in cell.line. Overwrite the vector with the number of missing values with the p-values in a suitable manner. (b) Download GDS711 and repeat the above using ANOVA p-values with the covariate disease.state to indicate the groups. (c) Download GDS2126 and repeat the above using ANOVA p-values with the covariate disease.state to indicate the groups. (d) Compute the symbols of the twenty best genes in the sense of having smallest summed p-values. (e) Summarize information of the twenty best genes in an HTML table. Does p53 play a role in the path way of the best gene? 9. Analysis of genes from a GO search. (a) Select the patients on the covariate mol.biol with values ALL1/AF4, BCR/ABL, and NEG. (b) Collect the ANOVA p-values with contrast between NEG and ALL1/AF4, and between NEG and BCR/ABL. Report the number of signiﬁcant aﬀy ID’s and the total. Hint: Re-order the columns into ”NEG”, ”ALL1/AF4”, and ”BCR/ABL”. (c) Find the GO ID’s refereing to the term ”protein-tyrosine kinase” since it mediates many steps due to BCR/ABL translocation. (d) Select the aﬀy ID’s corresponding to the GO ID’s and report its number and the number of signiﬁcant genes. (e) Perform Fisher exact to test the odds ratio equal to one hypoth- esis. 116 CHAPTER 6. MICRO ARRAY ANALYSIS Chapter 7 Cluster Analysis and Trees Given the expression values of several genes, a problem which often arises is to ﬁnd genes which are similar or close. Genes with expressions in small distance may have similar functions and may be potentially interesting for further research. In order to discover genes which form a group there are sev- eral methods developed called cluster analysis. These methods are based on a distance function and an algorithm to join data points to clusters. The so- called single linkage cluster analysis is intuitively appealing and often applied in bioinformatics. By this method several clusters of genes can be discov- ered without specifying the number of clusters on beforehand. The latter is necessary for another method called k-means cluster analysis. Each analysis produces a tree which represents similar genes as close leaves and dissimilar ones on diﬀerent edges. An other measure to investigate similarity or dependency of pairs of gene expressions is the correlation coeﬃcient. Various examples of applications will be given. It prepares the way to searching a data set for directions of large variance. That is, since gene expression data sets tend to be large, it is of importance to have a method available which discovers important “directions” in the data. A frequently used method to ﬁnd such directions is that by principal components analysis. Its basic properties will be explained as well as how it can be applied in combination with cluster analysis. In applications where it is diﬃcult to formulate distributional assumptions of the statistic it may still be of importance to construct a conﬁdence interval. It will be illustrated by several examples how the bootstrap can be applied to construct 95% conﬁdence intervals. Many examples are given to clarify the application of cluster analysis and principal components analysis. 117 118 CHAPTER 7. CLUSTER ANALYSIS AND TREES 7.1 Distance The concept of distance plays a crucial role in all types of cluster analysis. For real numbers a and b a distance function d is deﬁned as the absolute value of their diﬀerence d(a, b) = |a −b| = _ (a −b) 2 . The properties of a distance function should be in line with our intuition. That is, if a = b, then d(a, a) = 0 and if a = b, then d(a, b) > 0. Hence, the distance measure should be deﬁnitive in the sense that d(a, b) = 0 if and only if a = b. Since the square is symmetric, it follows that d(a, b) = |a −b| = _ (a −b) 2 = _ (b −a) 2 = |b −a| = d(b, a). In other words, d(a, b) = d(b, a), the distance between a and b equals that between b and a. Furthermore, it holds for all points c between a and b that d(a, b) = d(a, c) +d(c, b). For all points c not between a and b, it follows that d(a, b) < d(a, c) + d(c, b). The latter two notions can be summarized by the so-called triangle inequality. That is, for all real c it holds that d(a, b) ≤ d(a, c) + d(c, b). Directly going from a to b is shorter than via c. Finally, the distance between two points a and b should increase as these move further apart. Example 1. Let a = 1 and b = 3. Then, obviously, the distance d(1, 3) = 2. The number c = 2 is between a and b, so that d(1, 3) = 2 = 1 + 1 = d(1, 2) + d(2, 3) and the triangle inequality becomes an equality. For the situation where gene expression values for several patients are available, it is of importance to deﬁne a distance for vectors of gene expres- sions such as a = (a 1 , · · · , a n ) and b = (b 1 , · · · , b n ). We shall concentrate mainly on the Euclidian distance, which is deﬁned as the root of the sum of the squared diﬀerences d(a, b) = ¸ ¸ ¸ _ n i=1 (a i −b i ) 2 . 7.1. DISTANCE 119 The distance measure satisﬁes the above properties of deﬁniteness, symme- try, and triangle inequality. Although many other, but often highly similar, distance functions are available we shall mainly concentrate on Euclidian dis- tance because it is applied most frequently in bioinformatics. Example 2. Suppose that a = (a 1 , a 2 ) = (1, 1) and b = (b 1 , b 2 ) = (4, 5). Then d(a, b) = _ (a 1 −b 1 ) 2 + (a 2 −b 2 ) 2 = _ (1 −4) 2 + (1 −5) 2 = 9 + 16 = 5. Since the diﬀerences are squared it is immediate that d(a, b) = d(b, a), the distance from a to b equals that from b to a. For c = (c 1 , c 2 ) = (2, 2) we have that d(a, c) = 2, d(b, c) = 2 2 + 3 2 = 13. Hence, d(a, b) = 5 < 2 + 13 = d(a, c) + d(b, c), so that the triangle inequality is strict. This is in line with our intuitive idea that the road directly from a to b is shorter than from a to b via c. Example 3. To compute the Euclidian distance between two vectors one may use the following. > a <- c(1,1); b <- c(4,5) > sqrt(sum((a-b)^2)) [1] 5 Example 4. Distances between Cyclin gene expressions. By the build-in- function dist the Euclidian distance between two vectors of gene expression values can be computed. To select genes related to the biological term ”Cy- clin” and to compute the Euclidian distance between the gene expression values of the Golub et al. (1999) data, we may use the following. > library(multtest); data(golub) > index <- grep("Cyclin",golub.gnames[,2]) > golub.gnames[index,2] [1] "CCND2 Cyclin D2" [2] "CDK2 Cyclin-dependent kinase 2" [3] "CCND3 Cyclin D3" [4] "CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1)" [5] "CCNH Cyclin H" 120 CHAPTER 7. CLUSTER ANALYSIS AND TREES [6] "Cyclin-dependent kinase 4 (CDK4) gene" [7] "Cyclin G2 mRNA" [8] "Cyclin A1 mRNA" [9] "Cyclin-selective ubiquitin carrier protein mRNA" [10] "CDK6 Cyclin-dependent kinase 6" [11] "Cyclin G1 mRNA" [12] "CCNF Cyclin F" > dist.cyclin <- dist(golub[index,],method="euclidian") > diam <- as.matrix(dist.cyclin) > rownames(diam) <- colnames(diam) <- golub.gnames[index,3] > diam[1:5,1:5] D13639_at M68520_at M92287_at U09579_at U11791_at D13639_at 0.000000 8.821806 11.55349 10.056814 8.669112 M68520_at 8.821806 0.000000 11.70156 5.931260 2.934802 M92287_at 11.553494 11.701562 0.00000 11.991333 11.900558 U09579_at 10.056814 5.931260 11.99133 0.000000 5.698232 U11791_at 8.669112 2.934802 11.90056 5.698232 0.000000 By the grep function the order numbers of the genes with the phrase ”Cy- clin” in their names are assigned to the vector called index. The euclidian distances are assigned to the matrix called diam. Its diagonal has distances between identical genes which are, of course, zero. The distance between the ﬁrst (CCND2 Cyclin D2) and the third (CCND3 Cyclin D3) is relatively small, which is in line with the fact the these genes have related functions. Note, however, that there are genes with in smaller distance. Example 5. Finding the ten closest genes to a given one. After selecting certain genes it often happens that one wants to ﬁnd genes which are close to the selected ones. This can be done with the genefinder functionality by specifying either an index or a name (consistent with the geneNames of the exprSet). To ﬁnd genes from the ALL data (Chiaretti et al., 2004) close to the MME expression values of the probe with identiﬁer 1389_at, we may use the following. library("genefilter"); library("ALL"); data(ALL) closeto1389_at <- genefinder(ALL, "1389_at", 10, method = "euc") closeto1389_at[[1]]$indices
round(closeto1389_at[[1]]$dists,1) featureNames(ALL)[closeto1389_at[[1]]$indices]
7.2. TWO TYPES OF CLUSTER ANALYSIS 121
The function genefilter produces a list from which the selected row num-
bers can be extracted as well as the probe names can be found.
1
If desired,
these can be used for further analysis. From the output it can be observed
that the gene expressions of row 2653 with probe identiﬁer 32629_f_at has
the smallest distance (12.6) to those of 1389_at.
7.2 Two types of Cluster Analysis
Some important types of cluster analysis are deﬁned and illustrated here.
A cluster I is simply a set of data points I = {x
i
}, where x
i
is the i-th vector
with gene expressions. In single linkage cluster analysis the distance between
clusters I and J is deﬁned as the smallest distance over all pairs of points of
the two clusters:
d(I, J) = min
i,j
{d(x
i
, x
j
, ) : x
i
in I and x
j
in J} .
Hence, the distance between the two clusters is the same as that of the nearest
neighbors. The algorithm of single linkage cluster analysis starts with creat-
ing as many clusters as data points. Next, the nearest two are determined
and these are merged into one cluster. Then the next two nearest clusters
are determined and merged into one cluster. This process continuous until
all points belong to one cluster.
Example 1. An explanatory example. To illustrate single linkage cluster
analysis suppose we the following ﬁve gene expressions g
1
= (1, 1), g
2
=
(1, 1.2), g
3
= (3, 2), g
4
= (3, 2.2), and g
5
= (5, 5), from two persons. The
expressions for each gene can be seen as coordinates on two perpendicular
axis p
1
and p
2
. The script below produces Figure 7.1 which illustrates the
idea. It computes the the distances between the genes and performs a single
names <- list(c("g1","g2","g3","g4","g5"),c("p1","p2"))
1
For information on lists, see Chapter 6 of the manual ”An Introduction to R”.
122 CHAPTER 7. CLUSTER ANALYSIS AND TREES
1 2 3 4 5
1
2
3
4
5
a1
a
2
x1
x2
x3
x4
x5
Figure 7.1: Plot of ﬁve points to
be clustered.
x
5
x
1
x
2
x
3
x
4
0
.
0
0
.
5
1
.
0
1
.
5
2
.
0
2
.
5
3
.
0
3
.
5
Cluster Dendrogram
hclust (*, "single")
dist(sl.clus.dat, method = "euclidian")
H
e
i
g
h
t
Figure 7.2: Tree of single linkage
cluster analysis.
sl.clus.dat <- matrix(c(1,1,1,1.1,3,2,3,2.3,5,5),ncol = 2,
byrow = TRUE,dimnames = names)
plot(sl.clus.dat,type="n", xlim=c(0,6), ylim=c(0,6))
text(sl.clus.dat,labels=row.names(sl.clus.dat))
> print(dist(sl.clus.dat,method="euclidian"),digits=3)
x1 x2 x3 x4
x2 0.10
x3 2.24 2.19
x4 2.39 2.33 0.30
x5 5.66 5.59 3.61 3.36
> sl.out<-hclust(dist(sl.clus.dat,method="euclidian"),method="single")
> plot(sl.out)
At the start each data point is seen as a separate cluster. Then the nearest
two points (genes) from the Euclidian distance matrix are g
1
and g
2
, having
d(g
1
, g
2
) = 0.10. These two data points are merged into one cluster, say
I = {g
1
, g
2
}. In Figure 7.2 this is illustrated by the horizontal line at height
0.10 in the tree. The other three data points g
3
, g
4
, g
5
are seen as three
diﬀerent clusters. Next, the minimal distance between clusters can be read
from the Euclidian distance matrix. Since the smallest is d(g
3
, g
4
) = 0.30,
the new cluster J = {g
3
, g
4
}, corresponding to the horizontal line at height
7.2. TWO TYPES OF CLUSTER ANALYSIS 123
0.30. Now there are three clusters, I, J, and K = {x
5
}. From the Euclidian
distance matrix, it can be observed that the distance between cluster I and
J is 2.19, see the corresponding horizontal line at this height. Hence, the
cluster I and J are merged into one. Finally, the distance between cluster
{g
1
, g
2
, g
3
, g
4
}, and the data point g
5
equals d(g
4
, g
5
) = 3.36, see the corre-
sponding horizontal line at this height.
9
2
1
1
5
3 5
4
1
8
8
1
4
2
0
1
0
1
7
1
1
1
6
1
9
7
1
3
6
1
2
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
1
.
0
Cluster Dendrogram
hclust (*, "single")
dist(rnorm(20, 0, 1), method = "euclidian")
H
e
i
g
h
t
Figure 7.3: Example of three with-
out clusters.
2
2
2
5
2
8
3
0
2
4
2
1
2
9
2
3
2
6
2
7
2 1 9 7
1
0 5 6 4 3 8
1
6
1
9
2
0 1
7
1
5
1
3
1
8
1
2
1
1
1
4
0
1
2
3
4
5
Cluster Dendrogram
hclust (*, "single")
dist(x, method = "euclidian")
H
e
i
g
h
t
Figure 7.4: Three clusters with dif-
ferent standard deviations.
Example 2. Relating data generation processes to cluster trees. It is of
importance to have some experience with data that does and does not con-
tain clusters. To illustrate this we perform single linkage cluster analysis on
twenty data points from the standard normal population.
sl.out<-hclust(dist(rnorm(20,0,1),method="euclidian"),method="single")
plot(sl.out)
From the resulting tree in Figure 7.3 one might get the impression that
there are ﬁve separate clusters in the data. Note, however, that there is no
underlying data generation process which produces separate clusters from
diﬀerent populations.
If, however, the data are generated by diﬀerent normal distributions,
then there are diﬀerent processes producing separate clusters. To illustrate
124 CHAPTER 7. CLUSTER ANALYSIS AND TREES
this, ten data points were sampled from the N(0, 0.1) population, ten from
N(3, 0.5), and ten from N(10, 1).
x <- c(rnorm(10,0,0.1),rnorm(10,3,0.5),rnorm(10,10,1.0))
plot(hclust(dist(x,method="euclidian"),method="single"))
plot(sl.out)
From the tree in Figure 7.4, it can be observed that there clearly exist three
clusters.
These examples illustrate that results from cluster analysis may very well
reveal population properties, but that some caution is indeed in order.
−0.5 0.0 0.5 1.0 1.5 2.0 2.5

1
0
1
2
CCND3 Cyclin D3
Z
y
x
i
n
ALL
AML
Figure 7.5: Plot of gene ”CCND3
Cyclin D3” and ”Zyxin” expres-
sions for ALL and AML patients.
2
1
3
5
2
9
1
6
2
0
1
0
1
9
5
1
2
2
6
4
1
3
1
5
2
2
2
4
1
3 6
1
1
2
3 8
2
7
7 9
2
5
1
7
1
8 2
1
4
3
4
3
0
3
7
3
2
3
6
3
8
3
3
2
8
3
1
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
1
.
0
1
.
2
Cluster Dendrogram
hclust (*, "single")
dist(clusdata, method = "euclidian")
H
e
i
g
h
t
diagram from gene ”CCND3 Cy-
clin D3” and ”Zyxin” expressions
values.
Example 3. Application to the Golub (1999) data. Recall that the ﬁrst
twenty seven patients belong to ALL and the remaining eleven to AML and
that we found earlier that the expression values of the genes ”CCND3 Cyclin
D3” and ”Zyxin” diﬀer between the patient groups ALL and AML. Figure
7.5 illustrates that the patient groups diﬀer with respect to gene expression
values. How to produce this plot and a single linkage cluster analysis is shown
by the script below.
7.2. TWO TYPES OF CLUSTER ANALYSIS 125
data(golub, package="multtest")
clusdata <- data.frame(golub[1042,],golub[2124,])
colnames(clusdata)<-c("CCND3 Cyclin D3","Zyxin")
gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
plot(clusdata, pch=as.numeric(gol.fac))
legend("topright",legend=c("ALL","AML"),pch=1:2)
plot(hclust(dist(clusdata,method="euclidian"),method="single"))
Figure 7.6 gives the tree from single linkage cluster analysis. Apart from
three expressions the tree shows two clusters corresponding to the two pa-
tient groups.
7.2.2 k-means
K-means cluster analysis is a popular method in bioinfomatics. It is deﬁned
by minimizing the within cluster sum of squares over K clusters. That is,
given the data points x
1
, · · · , x
n
the method seeks to minimize the function
K

k=1
n
k

i∈I
k
d
2
(x
i
, a
k
)
over all possible points a
1
, · · · a
K
. This is accomplished by an algorithm
(Hartigan & Wong, 1979) which starts by partitioning the data points into
K initial clusters, either at random or using some heuristic device. It then
computes the cluster means (step 1) and constructs a new partition by asso-
ciating each point with the closest cluster mean (step 2). The latter yields
new clusters of which the means are calculated (step 1). Then it constructs
a new partition by associating each point with the closest cluster mean (step
2). These two steps are repeated until convergence. The latter occurs when
the data points no longer change clusters. The iterative algorithm is fast
in the sense that it often converges in less iterations than the number of
points n, but it need not to attain the global minimum. For the optimal
points a
1
, · · · a
K
, it holds that these are equal to the mean per cluster, that
is a
k
= x
k
for each cluster k. When the data points are independent and
identically distributed, then the cluster means converge in probability to the
corresponding population means (Pollard, 1981).
126 CHAPTER 7. CLUSTER ANALYSIS AND TREES
−1 0 1 2 3

1
0
1
2
3
data[,1]
d
a
t
a
[
,
2
]
Figure 7.7: K-means cluster anal-
ysis.
x
5
x
1
x
2
x
3
x
4
0
.
0
0
.
5
1
.
0
1
.
5
2
.
0
2
.
5
3
.
0
3
.
5
Cluster Dendrogram
hclust (*, "single")
dist(sl.clus.dat, method = "euclidian")
H
e
i
g
h
t
Figure 7.8: Tree of single linkage
cluster analysis.
Example 1. Relating a data generation process to k-means cluster analysis.
To illustrate k-means cluster analysis we shall simulate gene expressions from
two diﬀerent normal populations. That is, we randomly take ﬁfty gene ex-
pressions for two persons from the N(0, 0.5) population and ﬁfty expressions
for two persons from the N(2, 0.5) population. The data points are collected
in two matrices of order ﬁfty by two which are placed one above the other.
On the total of one hundred data points a (k =)2-means cluster analysis is
performed.
> data <- rbind(matrix(rnorm(100,0,0.5), ncol = 2),
+ matrix(rnorm(100,2,0.5), ncol = 2))
> cl <- kmeans(data, 2)
K-means clustering with 2 clusters of sizes 50, 50
Cluster means:
[,1] [,2]
1 1.87304978 2.01940342
2 0.01720177 0.07320413
Clustering vector:
[1] 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
7.2. TWO TYPES OF CLUSTER ANALYSIS 127
[38] 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
[75] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
Within cluster sum of squares by cluster:
[1] 22.60733 20.54411
Available components:
[1] "cluster" "centers" "withinss" "size"
The output of k-means cluster analysis is assigned to a list called cl. Observe
that the cluster means are fairly close to the population means (0, 0) and
(2, 2). The Clustering vector indicates to which cluster each data point
(gene) belongs and that these correspond exactly to the two populations
from which the data are sampled. The variable cl$cluster contains cluster membership and can be used to specify the color of each data point a plot, as follows. > plot(data, col = cl$cluster)
> points(cl$centers, col = 1:2, pch = 8, cex=2) The data points are plotted in red and black circles and the cluster means by a star, see Figure 7.7. The sum of the within cluster sum of squares equals the minimal function value obtained by the algorithm. Before performing a k-means cluster analysis a plot from a single linkage cluster analysis may reveal the number of clusters. If the number of clusters is not at all clear, then it becomes questionable whether k-means is appropriate. For cases where the number of clusters is only moderately clear, the algorithm is more sensible to get stuck into a solution which is only locally optimal. Such solutions are of limited scientiﬁc value. To cope with the danger of suboptimal solutions one may simply run the algorithm repeatedly by using the nstart option. Another possibility is to use rational initial starting values for the cluster means. In particular, the sample means of potential clusters or the hypothesized population means can be used. > initial <- matrix(c(0,0,2,2), nrow = 2, ncol=2, byrow=TRUE) > cl<- kmeans(data, initial, nstart = 10) The so-called bootstrap (Efron, 1979) can be used to estimate 95% conﬁdence intervals around cluster means. The idea is to re-sample with replacement from the given sample one thousand times with replacement and to compute quantiles for the corresponding conﬁdence intervals. 128 CHAPTER 7. CLUSTER ANALYSIS AND TREES n <- 100; nboot<-1000 boot.cl <- matrix(0,nrow=nboot,ncol = 4) for (i in 1:nboot){ dat.star <- data[sample(1:n,replace=TRUE),] cl <- kmeans(dat.star, initial, nstart = 10) boot.cl[i,] <- c(cl$centers[1,],cl$centers[2,]) } > quantile(boot.cl[,1],c(0.025,0.975)) 2.5% 97.5% -0.1098886 0.1627979 > quantile(boot.cl[,2],c(0.025,0.975)) 2.5% 97.5% -0.04830563 0.19721732 > quantile(boot.cl[,3],c(0.025,0.975)) 2.5% 97.5% 1.730495 2.009014 > quantile(boot.cl[,4],c(0.025,0.975)) 2.5% 97.5% 1.898407 2.162019 From the bootstrap conﬁdence intervals the null hypothesis that the cluster population means are equal to (0, 0) and (2, 2) are accepted. Example 2. Application to the Golub (1999) data. In the above we found that the expression values of the genes ”CCND3 Cyclin D3” and ”Zyxin” are closely related to the distinction between ALL and AML. Hence, a 2-means cluster analysis of these gene expression values is appropriate here. > data <- data.frame(golub[1042,],golub[2124,]) > colnames(data)<-c("CCND3 Cyclin D3","Zyxin") > cl <- kmeans(data, 2,nstart = 10) > cl K-means clustering with 2 clusters of sizes 11, 27 Cluster means: CCND3 Cyclin D3 Zyxin 1 0.6355909 1.5866682 2 1.8938826 -0.2947926 7.2. TWO TYPES OF CLUSTER ANALYSIS 129 Clustering vector: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 27 28 29 30 31 32 33 34 35 36 37 38 2 1 1 1 1 1 1 1 1 1 1 1 Within cluster sum of squares by cluster: [1] 4.733248 19.842225 The two clusters discriminate exactly the ALL patients from the AML pa- tients. This can also be seen from Figure 7.9, where expression values of CCND3 Cyclin D3 are depicted on the horizontal axis and those of Zyxin on the vertical, and the ALL patients are in red and the AML patients in black. By the bootstrap the cluster means and their conﬁdence intervals can be estimated. > mean(data.frame(boot.cl)) X1 X2 X3 X4 0.6381860 1.5707477 1.8945878 -0.2989426 > quantile(boot.cl[,1],c(0.025,0.975)) 2.5% 97.5% 0.2548907 0.9835898 > quantile(boot.cl[,2],c(0.025,0.975)) 2.5% 97.5% 1.259608 1.800581 > quantile(boot.cl[,3],c(0.025,0.975)) 2.5% 97.5% 1.692813 2.092361 > quantile(boot.cl[,4],c(0.025,0.975)) 2.5% 97.5% -0.60802142 -0.02420802 The diﬀerence between the bootstrap means and the k-means from the orig- inal data gives an estimate of the estimation bias. It can be observed that the bias is small. The estimation is quite precise because the 95% bootstrap conﬁdence intervals are fairly small. 130 CHAPTER 7. CLUSTER ANALYSIS AND TREES −0.5 0.0 0.5 1.0 1.5 2.0 2.5 1 0 1 2 CCND3 Cyclin D3 Z y x i n Figure 7.9: Plot of kmeans (stars) cluster analysis on CCND3 Cyclin D3 and Zyxin discriminating between ALL (red) and AML (black) patients. 7.3 The correlation coeﬃcient A frequently used coeﬃcient to express the degree of linear relationship between two sets of gene expression values is the correlation coeﬃcient ρ. For two sequences of gene expressions such as x = (x 1 , · · · , x n ) and y = (y 1 , · · · , y n ), the correlation coeﬃcient ρ is estimated by ´ ρ = n i=1 (x i −x i )(y j −y j ) _ n i=1 (x i −x i ) 2 n i=1 (y j −y j ) 2 . The value of the correlation coeﬃcient is always between minus one and plus one. If the value is close to either of these values, then the variables are 7.3. THE CORRELATION COEFFICIENT 131 linearly related in the sense that the ﬁrst is a linear transformation of the second. That is, there are constants a and b such that ax i + b = y i for all i. By the function cor.test, the null hypothesis H 0 : ρ = 0 can be tested against the alternative H 0 : ρ = 0. Example 1. Teaching demonstration. To develop intuition with respect to the correlation coeﬃcient the function run.cor.examp(1000) of the TeachingDemos package is quite useful. It launches an interactive plot with 1000 data points on two random variables X and Y . When the correlation is near zero, then the data points are distributed along contours of circles. By moving the slider slowly from the left to the right it can be observed that all points are approximately on a straight line. If the sign of the correlation coeﬃcient is positive, then small/large values of X tend to go together with small/large values of Y . Example 2. Another teaching demonstration. By the function put.points.demo() it is possible to add and delete points to a plot which interactively re- computes the value for the correlation coeﬃcient. By ﬁrst creating a few points that lie together on a circle the corresponding correlation coeﬃcient will be near zero. By next adding one outlier, it can be observed that the correlation coeﬃcient changes to nearly ±1. This illustrates that the corre- lation coeﬃcient is not robust against outliers. Example 3. Application to the Golub (1999) data. We shall illustrate the correlation coeﬃcient by two sets of expression values of the MCM3 gene of the Golub et al. (1999) data. This gene encodes for highly conserved mini-chromosome maintenance proteins (MCM) which are involved in the initiation of eukaryotic genome replication. Here, we ﬁnd its row numbers, collect the gene expression value in vectors x and y, and compute the value of the correlation coeﬃcient by the function cor(x,y). > library(multtest); data(golub) > x <- golub[2289,]; y <- golub[2430,] > cor(x,y) [1] 0.6376217 The value is positive which means that larger values of x occur together with larger values of y and vice versa. This can also be observed by plot(x,y). By 132 CHAPTER 7. CLUSTER ANALYSIS AND TREES the function cor.test, the null hypothesis H 0 : ρ = 0 can be tested against the alternative H 0 : ρ = 0. It also estimates a 95% conﬁdence interval for ρ. > cor.test(x,y) Pearson’s product-moment correlation data: x and y t = 4.9662, df = 36, p-value = 1.666e-05 alternative hypothesis: true correlation is not equal to 0 95 percent confidence interval: 0.3993383 0.7952115 sample estimates: cor 0.6376217 The test is based on the normality assumption and prints therefore a t-value. Since the corresponding p-value is very small, we reject the null hypothesis of zero correlation. The left bound of the conﬁdence interval falls far to the right hand side of zero. Example 4. Conﬁdence interval by the bootstrap. Another method to con- struct a 95% conﬁdence interval is by the bootstrap. The idea (Efron, 1979) is to obtain thousand samples from the original sample with replacement and to compute the correlation coeﬃcient for each of these. This yields thousand coeﬃcients from which the quantiles for the 95% conﬁdence interval can be computed. > nboot <- 1000; boot.cor <- matrix(0,nrow=nboot,ncol = 1) > data <- matrix(c(x,y),ncol=2,byrow=FALSE) > for (i in 1:nboot){ + dat.star <- data[sample(1:nrow(data),replace=TRUE),] + boot.cor[i,] <- cor(dat.star)[2,1]} > mean(boot.cor) [1] 0.6534167 > quantile(boot.cor[,1],c(0.025,0.975)) 2.5% 97.5% 0.2207915 0.9204865 7.4. PRINCIPAL COMPONENTS ANALYSIS 133 Observe that the 95% conﬁdence interval is larger than that found by cor.test. This indicates that the assumption of normality may not be completely valid here. Since the conﬁdence interval does not contain zero, we reject the null- hypothesis of zero correlation. Example 5. Application to the Golub (1999) data. The ALL and AML patients of the Golub et al. (1999) data are indicated by zero and ones of the binary vector golub.cl. A manner to select genes it by the correlation of the expression values with this binary vector. Such can be computed by using the apply functionality. > library(multtest); data(golub) > corgol<- apply(golub, 1, function(x) cor(x,golub.cl)) > o <- order(corgol) By golub.gnames[o[3041:3051],2] it can be seen that various of these genes seem indeed to have important cell functions referred to by Golub et al. (1999). In particular, Interleukin 8 is recently related to inﬂammatory cytokine production in myeloid cells (Tessarz et al., 2007). 7.4 Principal Components Analysis To make the basic ideas behind principal components analysis explicit, it is wise to start with a small artiﬁcial example. Suppose that for six genes the standardized expression values on two patients (variables) became available as these are given in Table 7.1. The data are collected in a 6 by 2 data matrix Z, where e.g. element z 21 is expression value -0.40 which belongs to the second gene of the ﬁrst patient. The whole idea of principal components analysis is to ﬁnd new directions in the data along which there is maximal variation. A direction is deﬁned as a linear combination Zk of the data Z by a vector k with weights. The i-th element of the linear combination is the weighted sum 2 j=1 z ij k j . The direc- tion of maximal variation is deﬁned as the linear combination with maximal variance. To ﬁnd this direction the correlation matrix plays an important role. The latter contains the correlations between each pair of patients (vari- ables). In our case correlations between the columns (patients) in Table 7.1 134 CHAPTER 7. CLUSTER ANALYSIS AND TREES Table 7.1: Data set for principal components analysis. Var 1 Var 2 gene 1 1.63 1.22 gene 2 −0.40 0.79 gene 3 0.93 0.97 gene 4 −1.38 −1.08 gene 5 −0.17 −0.96 gene 6 −0.61 −0.93 can be placed in a matrix R, which has ones on the diagonal and the value 0.8 elsewhere. To illustrate a direction let’s try the linear combination k = (2, 1) 2 of the sample correlation matrix R. This gives Rk = _ 1 0.8 0.8 1 _ _ 2 1 _ = _ 2.8 2.6 _ . Both vectors k and Rk can be plotted in the xy-plane. The vector (2, 1) is plotted by drawing an arrow from (0,0) to the point with x = 2 and y = 1. This is done completely similar for (2.8, 2.6) in Figure 7.10. It can be observed that the two vectors (arrows) do not fall on the same line and therefore have diﬀerent directions. The crux of principal components analysis is that a linear combination with the same direction as the weights represent the direction of maximum variation. Such is the case if Rk diﬀers from k only by a constant of multiplication, that is there exists a constant d such that Rk = dk. We shall determine such a constant by ﬁnding the weights vector ﬁrst. To do so observe from our correlations matrix that the sum of both rows equals 1.8. Taking k = (1, 1) yields Rk = _ 1 0.8 0.8 1 _ _ 1 1 _ = _ 1.8 1.8 _ = 1.8 _ 1 1 _ = 1.8k. So that we obtain d = 1.8. A similar result follows by observing that the diﬀerences per row are equal in absolute value. That is, taking k = (1, −1) 2 For the sake of simple notation we shall not use the transposition operator T to indicate rows. 7.4. PRINCIPAL COMPONENTS ANALYSIS 135 yields Rk = _ 1 0.8 0.8 1 _ _ 1 −1 _ = _ 0.2 −0.2 _ = 0.2 _ 1 −1 _ = 0.2k. A vector k for which Rk = dk holds is called an eigenvector corresponding to the eigenvalue d. Eigenvectors are often re-scaled by dividing by their Euclid- ian length. Since the Euclidian length of (1, 1) is 1 2 + 1 2 = 2, we obtain the new eigenvector k 1 = (1/ 2, 1/ 2) ≈ (0.71, 0.71). Since the length of eigenvector (1, −1) also equals 2 the re-scaled second eigenvector equals k 2 = (1/ 2, −1/ 2) ≈ (0.71, −0.71). Now the ﬁrst principal component is deﬁned as Zk 1 and the second as Zk 2 . In practical applications the actual computation of eigenvectors and eigenvalues is performed by well-designed numerical methods (Golub & Van Loan, 1983). 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 V[,1] V [ , 2 ] Figure 7.10: Vectors of linear com- binations. −2 −1 0 1 2 2 1 0 1 2 Z[,1] Z [ , 2 ] Figure 7.11: First principal com- ponent with projections of data. Example 1. Using R on the above data. It is convenient to store the data of the ﬁrst two columns of Table 7.1 as a matrix object called Z. The correlations matrix can be computed by the built-in-function cor and the eigenvectors and eigenvalues by the built-in-function eigen, as follows. Z <- matrix(c( 1.63, 1.22, -0.40, 0.79, 0.93, 0.97, -1.38, 136 CHAPTER 7. CLUSTER ANALYSIS AND TREES -1.08, -0.17, -0.96, -0.61, -0.93), nrow=6, byrow=TRUE) K <- eigen(cor(Z)) The output is stored as an object called K which can be printed to the screen in two digits. > print(K,digits=2)$values
[1] 1.8 0.2
$vectors [,1] [,2] [1,] 0.71 0.71 [2,] 0.71 -0.71 The eigenvalues are assigned to K$values and the eigenvectors are the columns
of K$vectors. To compute the principal components we use the matrix mul- tiplication operator %*%. Then the ﬁrst principal component is deﬁned as the linear combination of the data with the ﬁrst eigenvector, Z%*%K$vec[,1]. To
print the scores on the ﬁrst and the second principal component one can use
the following.
> print(Z %*% K$vec, digits=2) [,1] [,2] [1,] 2.02 0.290 [2,] 0.28 -0.841 [3,] 1.34 -0.028 [4,] -1.74 -0.212 [5,] -0.80 0.559 [6,] -1.09 0.226 To illustrate the ﬁrst principal component the six data points from the Z matrix are plotted as small circles in Figure 7.11. Gene 1, for instance, has x coordinate 1.63 and y coordinate 1.22 and appears therefore in the right upper corner. A convenient manner to perform principal components analysis is by using the built-in-function princomp, as follows. pca <- princomp(Z, center = TRUE, cor=TRUE, scores=TRUE) pca$scores
7.4. PRINCIPAL COMPONENTS ANALYSIS 137
The scores are the component scores and the loadings from princomp are
the eigenvectors.
The eigenvalues represent an amount of variance related to the compo-
nent. In the previous example the ﬁrst component has variance 1.8 and the
second 0.2, so that the ﬁrst represents 1.8/2 = 0.9 or 90% of the variance. On
the basis of the eigenvalues the number of interesting directions in the data
can be evaluated by two rules of thumb. The ﬁrst is that each eigenvalue
should represent more variance than that of any of the observed variables.
The second is the so-called elbow rule saying that when the ﬁrst few eigen-
values are large and the remaining considerably smaller, then the ﬁrst few
are the most interesting.
Principal components analysis is a descriptive method to analyze depen-
dencies (correlations) between variables. If there are a few large eigenvalues,
then there are equally many directions in the data which summarize the
most important variation among the gene expressions. Then it may be use-
ful to explore simultaneously a two dimensional visualization of the genes
and the patients. Furthermore, it can be rewarding to study the weights of
the eigenvectors because these may reveal a structure in the data otherwise
gone unnoticed. Finally, the principal components contain less (measure-
ment) error than the individual variables. For this reason, cluster analysis
on the values on the principal components may be useful.
Example 2. Application to the Golub (1999) data. The ﬁrst ﬁve eigenvalues
from the correlation matrix of golub can be printed by the following.
> eigen(cor(golub))$values[1:5] [1] 25.4382629 2.0757158 1.2484411 1.0713373 0.7365232 Because the eigenvalues are arranged in decreasing order the sixth to the 38th are smaller than one. Reason for which these will be neglected. The ﬁrst eigenvalue is by far the largest, indicating that the persons are dependent to a large extent. Applying the previous bootstrap methods to estimate 95% conﬁdence intervals for the eigenvalues we obtain the following intervals. data <- golub; p <- ncol(data); n <- nrow(data) ; nboot<-1000 eigenvalues <- array(dim=c(nboot,p)) for (i in 1:nboot){dat.star <- data[sample(1:n,replace=TRUE),] eigenvalues[i,] <- eigen(cor(dat.star))$values}
138 CHAPTER 7. CLUSTER ANALYSIS AND TREES
> for (j in 1:p) print(quantile(eigenvalues[,j],c(0.025,0.975)))
2.5% 97.5%
for (j in 1:5) cat(j,as.numeric(quantile(eigenvalues[,j],
+ c(0.025,0.975))),"\n" )
1 24.83581 26.00646
2 1.920871 2.258030
3 1.145990 1.386252
4 0.9917813 1.154291
5 0.6853702 0.7995948
The cat function allows for much control in printing. The null hypothesis
of eigenvalue being equal to one is accepted for the fourth component and
rejected for the ﬁrst three and the ﬁfth. Thus the fourth represents less
variance than an individual variable, reason for which it is neglected.
The percentages of variance explained by the ﬁrst two components can be
computed by sum(eigen(cor(golub))$values[1:2])/38*100, which yields the amount 72.4052%. Thus the ﬁrst two components represent more than 72% of the variance in the data. Hence, the data allow for a reduction in dimensions from thirthy eight to two. −10 −5 0 5 10 15 1 0 5 0 5 1 0 X1 X 2 68 182 313 504 792808 885 892 893 1101 1616 1737 1754 1756 1798 1882 1910 1911 2233 2321 2350 2397 2459 2611 2653 2673 2749 2761 2874 450 849 316 2289 2430 Figure 7.12: Scatter plot of se- lected genes with row labels on the ﬁrst two principal components. 1 9 1 0 2 8 7 4 2 3 5 0 1 7 3 7 1 9 1 1 2 7 4 9 2 7 6 1 7 9 2 2 3 2 1 6 8 8 0 8 2 6 1 1 4 5 0 5 0 4 3 1 3 1 7 5 6 8 9 3 1 8 2 2 6 5 3 2 2 8 9 2 4 3 0 3 1 6 1 6 1 6 1 1 0 1 2 6 7 3 2 4 5 9 8 9 2 1 8 8 2 8 8 5 8 4 9 2 3 9 7 1 7 5 4 1 7 9 8 2 2 3 3 0 . 0 0 . 5 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 Cluster Dendrogram hclust (*, "single") dist(leu, method = "euclidian") H e i g h t Figure 7.13: Single linkage cluster diagram of selected gene expres- sion values. It can be checked that all correlations between the patients are positive. 7.4. PRINCIPAL COMPONENTS ANALYSIS 139 This implies that large expression values on gene i co-vary positively with large deviations of gene j. The positivity of the correlations also implies that the weights of the ﬁrst eigenvector have the same sign, so that these can be taken to be positive for all patients (Horn & Johnson, 1985). Unfortunately, this is not automatic in R so that caution is in order with respect to inter- pretation of the components. By using -eigen(cor(golub))$vec[,1:2] to
print the weights to the screen it can be observed that those that correspond
to the ﬁrst component are positive. All weights of the ﬁrst eigenvector are
positive and have very similar size (all are between 0.13 and 0.17). Thus the
ﬁrst component is almost equal to the sum of the variables (the correlation
equals 0.9999). The weights of the second component have a very interesting
pattern. Namely, almost all of the ﬁrst 27 weights are positive and the last 11
weights are negative. Thus the second component contrasts the ALL patients
with the AML patients. By contrasting ALL patients with AML patients a
second to the largest amount of variance is explained in the data. Hence,
the AML-ALL distinction is discovered by the second component, which is
in line with ﬁndings of Golub et al. (1999).
Obviously the genes with the largest expression values from the ﬁrst com-
ponent can be printed. We shall, however, concentrate on the second compo-
nent because it appears to be more directly related to the research intentions
of Golub et. al. (1999). The ﬁrst and the last ten gene names with respect
to the values on the second component can be printed by the following.
> pca <- princomp(golub, center = TRUE, cor=TRUE, scores=TRUE)
> o <- order(pca$scores[,2]) > golub.gnames[o[1:10],2] > golub.gnames[o[3041:3051],2] Many of these genes are related to leukemia (Golub, et al., 1999). Example 3. Biplot. A useful manner to plot both genes (cases) and patients (variables) is the biplot, which is based on a two-dimensional approximation of the data very similar to principal components analysis. Here, we illustrate how it can be combined with principal components analysis. > biplot(princomp(data,cor=TRUE),pc.biplot=TRUE,cex=0.5,expand=0.8) The resulting plot is given by Figure 7.14. The left and bottom axis refer to the component scores and the top and right to the patient scores, which 140 CHAPTER 7. CLUSTER ANALYSIS AND TREES are scaled to unit length by the speciﬁcation cor. It can be seen that the patients are clearly divided in two groups corresponding to ALL and AML. Example 4. Critical for S-phase. Golub et al. (1999) mention that among genes which are useful for tumor class prediction there are genes that encode for proteins critical for S-phase cell cycle progression such as Cyclin D3, Op18, and MCM3. We select genes which carry ”CD”, ”Op”, or ”MCM” in their names and collect the corresponding row numbers. data(golub, package = "multtest") factor <- factor(golub.cl) o1 <- grep("CD",golub.gnames[,2]) o2 <- grep("Op",golub.gnames[,2]) o3 <- grep("MCM",golub.gnames[,2]) o <- c(o1,o2,o3) This yields 110 genes. In order to select those that do have an experimental eﬀect, we use a two-sample t-test. pt <- apply(golub, 1, function(x) t.test(x ~ gol.fac)$p.value)
oo <- o[pt[o]<0.01]
This yields 34 genes, of which the row numbers are selected in the vector
oo. In order to identify genes in directions of large variation we use the scores
on the ﬁrst two principal components.
Z <- as.matrix(scale(golub, center = TRUE, scale = TRUE))
K <- eigen(cor(Z))
P <- Z %*% -K$vec[,1:2] leu <- data.frame(P[oo,], row.names= oo) attach(leu) The scores on the ﬁrst two principal components of the selected genes are stored in the data frame leu. From the plotted component scores in Figure 7.12, it seems that there are several sub-clusters of genes. The genes that belong to these clusters can be identiﬁed by hiearchical cluster analysis. cl <- hclust(dist(leu,method="euclidian"),method="single") plot(cl) 7.5. OVERVIEW AND CONCLUDING REMARKS 141 From the tree (dendrogram) in Figure 7.13 various clusters of genes are apparent that also appear in Figure 7.12. 3 The ordered genes can be obtained from the object cl as follows. > a <- as.integer(rownames(leu)[cl$order])
> for (i in 1:length(a)) cat(a[i],golub.gnames[a[i],2],"\n")
1910 FCGR2B Fc fragment of IgG, low affinity IIb, receptor for (CD32)
2874 GB DEF = Fas (Apo-1, CD95)
The cluster with rows 504, 313, 1756, and 893 consists of antigenes. The
genes MCM3 Minichromosome maintenance deﬁcient (S. cerevisiae) 3 with
row numbers 2289 and 2430 appear adjacent to each other. This illustrates
that genes with similar functions may indeed be close with respect to their
gene expression values.
7.5 Overview and concluding remarks
Single linkage cluster analysis can be applied to explore for groups in a set of
gene expressions. When groups are present a k-means cluster analysis can be
applied in combination with the bootstrap to estimate conﬁdence intervals
for the cluster means.
The correlation coeﬃcient measures the degree of dependency between
pairs of gene expression values. It can also be used to ﬁnd gene expressions
which are highly dependent with a phenotypical variable. It is reassuring to
ﬁnd in applications that the conﬁdence interval for a correlation coeﬃcient
is small.
Principal components analysis is very useful for ﬁnding directions in the
data where the gene expression values vary maximally, see Jolliﬀe (2002) for
a complete treatment of the principal component analysis. When these di-
rections can be represented well by the ﬁrst two components a biplot helps to
simultaneously visualize genes and patients. Principal components analysis
can be useful in identifying clusters of genes in a lower dimensional space.
3
Unfortunately, some row numbers of genes are less readable because the points are
very close.
142 CHAPTER 7. CLUSTER ANALYSIS AND TREES
7.6 Exercises
1. Cluster analysis on the ”Zyxin” expression values of the Golub et al.
(1999) data.
(a) Produce a chatter plot of the gene expression values using showing
diﬀerent symbols for the two groups.
(b) Use single linkage cluster analysis to see whether the three indi-
cates two diﬀerent groups.
(c) Use k-means cluster analysis. Are the two clusters according to
the diagnosis of the patient groups?
(d) Perform a bootstrap on the cluster means. You will have to modify
the code here and there. Do the conﬁdence intervals for the cluster
means overlap?
2. Close to CCND3 Cyclin D3. Recall that we did various analysis on
the expression data of the CCND3 Cyclin D3 gene of the Golub (1999)
data.
(a) Use genefilter to ﬁnd the ten closed genes to the expression
values of CCND3 Cyclin D3. Give their probe as well as their
biological names.
(b) Produce of combined boxplot separately for the ALL and the AML
expression values. Compare it with that on the basis of CCND3
Cyclin D3 and comment of the similarities.
(c) Compare the smallest distances with those among the Cyclin genes
computed above. What is your conclusion?
3. MCM3. In the example on MCM3 a plot shows that there is an outlier.
(a) Plot the data and invent a manner to ﬁnd the row number of the
outlier.
(b) Remove the outlier, test the correlation coeﬃcient. Compare the
results to those above.
(c) Perform the bootstrap to construct a conﬁdence interval.
4. Cluster analysis on part of Golub data.
7.6. EXERCISES 143
(a) Select the oncogenes from the Golub data and plot the tree from
(b) Do you observe meaningful clusters.
(c) Select the antigenes and answer the same questions.
(d) select the receptor genes and answer the same questions.
5. Principal Components Analysis on part of the ALL data.
(a) Construct an expression set with the patients with B-cell in stage
B1, B2, and B3. Compute the corresponding ANOVA p-values
of all gene expressions. Construct the expression set with the p-
values smaller than 0.001. Report the dimensionality of the data
matrix with gene expressions.
(b) Are the correlations between the patients positive?
(c) Compute the eigenvalues of the correlation matrix. Report the
largest ﬁve. Are the ﬁrst three larger than one?
(d) Program a bootstrap of the largest ﬁve eigenvalues. Report the
bootstrap 95% conﬁdence intervals and draw relevant conclusions.
(e) Plot the genes in a plot of the ﬁrst two principal components.
6. Some correlation matrices.
_
1 −0.8
−0.8 1
_
,
_
_
1 0.8 0.8
0.8 1 0.8
0.8 0.8 1
_
_
,
_
_
1 −0.5 −0.5
−0.5 1 −0.5
−0.5 −0.5 1
_
_
,
(a) Verify that the eigenvalues of the matrices are 1.8, 0.2, 2.6,
0.2, 0.2, and 1.500000e+00, 1.500000e+00, -7.644529e-17.
(b) How much variance represents the ﬁrst component corresponding
to the second matrix?
(c) Verify that the ﬁrst eigen vector of the second correlation matrix
has identical signs.
144 CHAPTER 7. CLUSTER ANALYSIS AND TREES
−3 −2 −1 0 1 2

3

2

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Figure 7.14: Biplot of selected genes from the golub data.
Chapter 8
Classiﬁcation Methods
In medical settings groups of patients are often diagnosed into classes corre-
sponding to types of diseases. In bioinformatics the question arises whether
the diagnosis of a patient can be predicted by gene expression values? Re-
lated is the question which genes play an important role in the prediction of
class membership. A similar question is the prediction of micro RNA’s from
values of folding energy. More generally, for objects like proteins, mRNA’s,
or microRNA’s it may be of importance to classify these on the basis of
certain measurements.
Many classiﬁcation methods have been developed for various scientiﬁc
purposes. In bioinformatics, methods such as recursive partitioning, support
vector machine and neural network are frequently applied to solve classiﬁca-
tion problems.
In this chapter you learn what recursive partitioning is and how to use it.
To evaluate the quality of prediction the fundamental concepts of sensitivity
and speciﬁcity are frequently used. The speciﬁcity can be summarized in a
single number by the area under the curve of a receiver operator curve. This
will be explained and illustrated. Two other methods to predict disease class
from gene expression data are the support vector machine and the neural
network. It will brieﬂy be explained what these methods are about and how
these can be applied. A validation set will be used to evaluate the predictive
accuracy.
145
146 CHAPTER 8. CLASSIFICATION METHODS
8.1 Classiﬁcation of microRNA
The subject of making a correct medical diagnosis is highly similar to that
of correctly classifying microRNA.
Example 1. Classiﬁcation of Micro RNA. MicroRNA are small RNA molecules
with important functions in cell growth and disease development. In order
to identify microRNA’s from arbitrary sequences its characterizing proper-
ties are used to distinguish non-microRNA from microRNA molecules. One
of these properties is that microRNA’s have the capacity to fold in a cer-
tain hairpin type of structure. Such a structure typically exhibits a small
minimum folding energy (Zuker, 2003; Zuker & Stiegler, 1981). This prop-
erty can be used as a test to discriminate microRNA’s from non-microRNA’s
(Bonnet, et al., 2004), as follows. Given a set of 3424 diﬀerent microRNA’s
the minimum folding energy was computed for each of these. Next, for each
microRNA the order of the nucleotides was shuﬄed with replacement 1000
times. This yielded per microRNA 1000 diﬀerently shuﬄed sequences of nu-
cleotides for which the minimum folding energy is computed.
1
Per microRNA
the 1001 energy values were arranged to have increasing order, similar as for
empirical distributions in the previous chapter. Then the number of mini-
mum folding energies below that of the original microRNA is counted and
divided by 1001 as the p-value. If the minimum folding energie of the original
microRNA is the smallest, then the empirical p-value is zero. This proce-
dure yielded a total of 3424 p-values. The number of sequences with p-values
below the threshold value 0.01 is given in Table 8.1. The same procedure is
conducted for non-microRNA molecules which were taken as sequences with
similar length and nucleotide percentages.
Table 8.1: Frequencies empirical p-values lower than or equal to 0.01.
test positive test negative total
p ≤ 0.01 p > 0.01
microRNA 2973 451 3424
non microRNA 33 3391 3424
total 3006 3842 6848
1
I am obliged to Sven Warris for computing the minimum energy values.
8.2. ROC TYPES OF CURVES 147
From the frequency Table 8.1, the sensitivity, the speciﬁcity, and the
predictive power can be computed in order to evaluate the quality of the
test. The sensitivity is the probability that the test is positive given that the
sequence is a microRNA (true positive). Thus
sensitivity = P(true positive) = P(test positive|microRNA) =
2973
3424
= 0.8682.
The speciﬁcity is the probability that the test is negative given that the
sequence is not a microRNA (true negative). Thus
speciﬁcity = P(true negative) = P(test negative|no microRNA) =
3391
3424
= 0.9903.
For practical applications of a test the predictive power is of crucial impor-
tance. In particular, the predictive value positive is the probability that the
sequence is a microRNA given that the test is positive. That is,
Predictive value positive = PV
+
= P(microRNA|test positive) =
2973
3006
= 0.9890
Thus when the test is positive we are 98.90% certain that the sequence is
indeed a microRNA. The predictive value negative is the probability that the
sequence is not a microRNA given that the test is negative.
Predictive value negative = PV

= P(no microRNA|test negative) =
3391
3842
= 0.8826.
Thus when the test is negative we are 88.26% certain that the sequence
is not a microRNA. From the estimated conditional probabilities it can be
concluded that the test performs quite well in discriminating between mi-
croRNA’s from non-microRNA’s.
8.2 ROC types of curves
In Chapter 2 we have observed with respect to the Golub et al. (1999) data
that the expression values of gene CCND3 Cyclin D3 tend to be greater for
ALL patients. We may therefore use these as a test for predicting ALL using
a certain cutoﬀ value. In particular, for gene expression values larger than a
cutoﬀ we declare the test “positive” in the sense of indicating ALL. By doing
148 CHAPTER 8. CLASSIFICATION METHODS
so the corresponding true and false positives can be computed for each cutoﬀ
value. To brieﬂy indicate the origin of the terminology, imagine that the test
characteristic (ROC) is a curve where the false positive rates are depicted
horizontally and the true positive rates vertically. The larger the area under
the ROC curve, the better the test is because then low false positive rates
go together with large true positive rates.
2
These ideas are illustrated by
several examples.
Example 1. For the sake of illustration we consider the prediction of ALL
from the expression values for gene CCND3 Cyclin D3 from Golub et al.
(1999) in row 1042 of the matrix golub. Now consider cutoﬀ point 1.27. For
such a cutoﬀ point we can produce a table with TRUE/FALSE frequencies
of predicting ALL/not ALL.
> data(golub, package = "multtest")
> gol.true <- factor(golub.cl,levels=0:1,labels= c(" ALL","not ALL"))
> gol.pred <- factor(golub[1042,]>1.27,levels=c("TRUE","FALSE"),
labels=c("ALL","notALL"))
> table(gol.pred,gol.true)
gol.true
gol.pred ALL not ALL
ALL 25 1
notALL 2 10
There are 25 ALL patients with expression values greater than or equal to
1.27, so that the true positive rate is 25/27=0.93. For this cutoﬀ value there
is one false positive because one patient without ALL has a score larger than
1.27. Hence, the false positive rate is 1/11 = 0.09.
Example 2. The expression values for gene CCND3 Cyclin D3 from the
Golub et al. (1999) data are sorted in decreasing order, see Table 8.2. The
procedure to draw the ROC curve starts with cutoﬀ point inﬁnity. Obviously,
there are no expression values equal to inﬁnity, so there is no patient tested
positive. Next, the cut oﬀ point 2.77 is taken and values greater than or
equal to 2.77 are tested as positive. This yields one true positive implying a
2
More detailed information can be obtained from a wikipedia search using ”ROC
curve”.
8.2. ROC TYPES OF CURVES 149
true positive rate of 1/27, see second row of Table 8.2. For this cutoﬀ value
there are no negatives so that the false positive rate is zero.
Now consider cutoﬀ point 1.52. There are 22 ALL patients with expres-
sion values greater than or equal to 1.52, so that the true positive rate is
22/27=0.81. For this cutoﬀ value there are no false positives because all pa-
tients without ALL have expression values lower than 1.51. Hence, the false
positive rate is 0 and the true positive rate is 0.81. To indicate this there is
a vertical line drawn in the ROC curve from point (0, 0) to point (0, 0.81)
in Figure 8.1. Now consider the next cutoﬀ point 1.45. There are 22 ALL
patients with expression values greater than or equal to 1.45, so that the
true positive rate is again 22/27=0.81. However, there is one patient with-
out ALL having expression value 1.45, whom receives therefore a positive
test. Hence, the number of false positives increases from zero to one, which
implies a false positive rate of 1/11=0.09. In the ROC curve this is indicated
by point (0.09, 0.81) and the horizontal line from (0, 0.81) to (0.09, 0.81), see
Figure 8.1.
This process goes on (see Table 8.2) until the smallest data point -0.74 is
taken as cutoﬀ point. For this point all patients are tested positive, so that
the false positive rate is 11/11 and the true positive rate is 27/27. This is
indicated by the end point (1, 1) in the plot at the top on the right hand side.
False positive rate
T
r
u
e

p
o
s
i
t
i
v
e

r
a
t
e
0.0 0.2 0.4 0.6 0.8 1.0
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
1
.
0
Figure 8.1: ROC plot for expres-
sion values of CCND3 Cyclin D3.
False positive rate
T
r
u
e

p
o
s
i
t
i
v
e

r
a
t
e
0.0 0.2 0.4 0.6 0.8 1.0
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
1
.
0
Figure 8.2: ROC plot for expres-
sion values of gene Gdf5.
150 CHAPTER 8. CLASSIFICATION METHODS
It is obviously helpful to use a computer for producing an ROC such as
in Figure 8.1. To do so we construct an appropriate factor with the value
TRUE for ALL and FALSE for not ALL gol.true and use functions from
the ROCR package.
library(ROCR)
gol.true <- factor(golub.cl,levels=0:1,labels= c("TRUE","FALSE"))
pred <- prediction(golub[1042,], gol.true)
perf <- performance(pred, "tpr", "fpr" )
plot(perf)
It seems clear that the expression values are better in testing for ALL when
the curve is very steep in the beginning and attains its maximum value soon.
In such a case the true positive rate is large for a small false positive rate. A
manner to express the predictive accuracy of a test in a single number is by
the area under the curve. Using the function performance(pred,"auc") we
obtain that the area under the curve is 0.96, which is large. Hence, the ex-
pression values of CCND3 Cyclin D3 are suitable for discrimination between
ALL and not ALL (AML). The ROC curve for the expression values of gene
Gdf5 is given by Figure 8.2. It can be observed that the true positive rate
is much lower as one moves on the horizontal axis from the left to the right.
This corresponds to the area under the curve of 0.35, which is small. This
illustrates that genes may express large diﬀerences with respect to prediction
of the disease status of patients.
In practical applications one is often interested in a single optimal cut-oﬀ
value and in combining several predictors in a decision scheme.
8.3 Classiﬁcation trees
The purpose of classiﬁcation is to allocate organisms into classes on the ba-
sis of measurements on attributes. For instance, in case of the Golub et al.
(1999) data the organisms are 38 patients which have measurements on 3051
genes. The classes consist of diagnosis of patients into the ALL class (27
patients) and the AML class (11 patients). A tree model resembles that of
a linear model, where the criterion is the factor indicating class membership
and the predictor variables are the gene expression values. In case of, for in-
stance, the Golub et al. (1999) data the gene expression values {x
1
, · · · , x
38
}
8.3. CLASSIFICATION TREES 151
can serve as predictors to form a decision tree. For instance, if x
j
< t, then
patient j is AML, and otherwise if x
j
≥ t, then patient j is ALL. Obvi-
ously, the threshold value t on which the decision is based should be optimal
given the predictor. Such can be estimated by a regression tree (Breiman
et al., 1984; Chambers & Hastie, 1992; Venables, & Ripley, 2000), which is
implemented in the rpart package (Therneau & Atkinson, 1997).
A training set is used to estimate the threshold values that construct the
tree. When many predictor variables are involved, 3051 for instance, then we
have a tremendous gene (variable) selection problem. The rpart package au-
tomatically selects genes which are important for classiﬁcation and neglects
others. A further problem is that of overﬁtting where additional nodes of a
tree are added to increase prediction accuracy. When such nodes are speciﬁc
for the training sample set, these can not be generalized to other samples
so that these are of limited scientiﬁc value. Prevention of such overﬁtting is
called pruning and is automatically done by the rpart function. Many basic
ideas are illustrated by an elementary example.
ALL1 ALL2 AML
0
1
2
3
4
Figure 8.3: Boxplot of expression
values of gene a for each leukemia
class.
genea< 0.9371
genea< 3.025
ALL1
10/0/0
ALL2
0/10/0
AML
0/0/10
Figure 8.4: Classiﬁcation tree for
gene for three classes of leukemia.
Example 1. Optimal gene expressions. Suppose microarray expres-
sion data are available with respect to patients suﬀering from three types of
152 CHAPTER 8. CLASSIFICATION METHODS
leukemia abbreviated as ALL1, ALL2, and AML. Gene A has expression val-
ues from the populations (patient groups) N(0, 0.5
2
) for ALL1, N(2, 0.5
2
) for
ALL2, and N(4, 0.5
2
) for AML. The script below generates thirty expression
values for gene A, the patients of the three disease classes, and the estimates
of the classiﬁcation tree.
set.seed(123); n<-10 ; sigma <- 0.5
fac <- factor(c(rep(1,n),rep(2,n),rep(3,n)))
levels(fac) <- c("ALL1","ALL2","AML")
geneA <- c(rnorm(10,0,sigma),rnorm(10,2,sigma),rnorm(10,4,sigma))
dat <- data.frame(fac,geneA)
library(rpart)
rp <- rpart(fac ~ geneA, method="class",data=dat)
plot(rp, branch=0,margin=0.1); text(rp, digits=3, use.n=TRUE)
From the boxplot in Figure 8.3 it can be observed that there is no overlap of
gene expressions between classes. This makes gene A an ideal predictor for
separating patients into classes. By the construction of the gene expression
values x
1
, · · · , x
30
we expect the following partition. If x
i
< 1, then ALL1,
if x
i
is in interval [1, 3], then ALL2, and if x
i
> 3, then AML. From the
estimated tree in Figure 8.4 it can be observed that the estimated splits are
close to our expectations: If x
i
< 0.971, then ALL1, if x
i
is in [0.9371, 3.025],
then ALL2, and if x
i
> 3.025, then AML. The tree consists of three leaves
(nodes) and two splits. The prediction of patients into the three classes per-
fectly matches the true disease status.
Obviously, such an ideal gene need not exist because the expression values
overlap between the disease classes. In such a case more genes may be used
to build the classiﬁcation tree.
Example 2. Gene selection. Another situation is where Gene A discrim-
inates between ALL and AML and Gene B between ALL1 patients and ALL2
or AML patients and Gene C does not discriminate at all. To simulate this
setting we generate expression values for Gene A from N(0, 0.5
2
) for both
ALL1 and ALL2, and from N(2, 0.5
2
) for AML patients. Next, we generate
expression values for Gene B from N(0, 0.5
2
) for ALL1 and from N(2, 0.5
2
)
for ALL2 and AML. Finally, we generate for Gene C from N(1, 0.5
2
) for
ALL1, ALL2, and AML. For this and for estimating the tree, we use the
following script.
8.3. CLASSIFICATION TREES 153
set.seed(123)
n<-10 ; sigma <- 0.5
fac <- factor(c(rep(1,n),rep(2,n),rep(3,n)))
levels(fac) <- c("ALL1","ALL2","AML")
geneA <- c(rnorm(20,0,sigma),rnorm(10,2,sigma))
geneB <- c(rnorm(10,0,sigma),rnorm(20,2,sigma))
geneC <- c(rnorm(30,1,sigma))
dat <- data.frame(fac,geneA,geneB,geneC)
library(rpart)
rp <- rpart(fac ~ geneA + geneB + geneC, method="class",data=dat)
Note the addition in the model notation for the rpart function.
3
It is con-
venient to collect the data in the form of a data frame.
4
From the boxplot in Figure 8.5 it can be seen that Gene A discriminates
well between ALL and AML, but not between ALL1 and ALL2. The expres-
sion values for Gene B discriminate well between ALL1 and ALL2, whereas
those of Gene C do not discriminate at all. The latter can also be seen from
the estimated tree in Figure 8.6, where Gene C plays no role at all. This il-
lustrates that rpart automatically selects the genes (variables) which play a
role in the classiﬁcation tree. Expression values on Gene A larger than 1.025
are predicted as AML and smaller ones as ALL. Expression values on Gene
B smaller than 0.9074 are predicted as ALL1 and larger as ALL2. Hence,
Gene A separates well within the ALL class.
Example 3. Prediction by CCND3 Cyclin D3 gene expression values.
From various visualizations and statistical testing in the previous chapters,
it can be conjectured that CCND3 Cyclin D3 gene expression values form a
suitable predictor for discriminating between ALL and AML patients. Note,
however, from Figures 2.2 and 8.7 that there is some overlap between the
expression values from the ALL and the AML patients, so that a perfect
classiﬁcation is not possible. By the function rpart the regression partition-
ing can be computed as follows.
> library(rpart);data(golub); library(multtest)
> gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
> gol.rp <- rpart(gol.fac ~ golub[1042,] , method="class")
3
See Chapter 11 of the manual ”An Introduction to R” for more on model notation.
4
See Chapter 6 of the manual ”An Introduction to R” for more on data frames.
154 CHAPTER 8. CLASSIFICATION METHODS
ALL1 ALL2 AML

1
0
1
2
Figure 8.5: Boxplot of expression
values of gene a for each leukemia
class.
genea< 1.025
geneb< 0.9074
ALL1
10/0/0
ALL2
0/10/0
AML
0/0/10
Figure 8.6: Classiﬁcation tree of
expression values from gene A,
B, and C for the classiﬁcation of
ALL1, ALL2, and AML patients.
> predictedclass <- predict(gol.rp, type="class")
> table(predictedclass, gol.fac)
gol.fac
predictedclass ALL AML
ALL 25 1
AML 2 10
Note that (25 + 10)/38 · 100% = 92.10% of the ALL/AML patients are cor-
rectly classiﬁed by gene CCND3 Cyclin D3. By the function predict(gol.rp,type="class")
the predictions from the regression tree of the patients in the two classes can
be obtained. The factor gol.fac contains the levels ALL and AML corre-
sponding to the diagnosis to be predicted. The predictor variable consists
of the expression values of gene CCND3 Cyclin D3. The output of recursive
partitioning is assigned to an object called gol.rp, a list from which fur-
ther information can be extracted by suitable functions. A summary can be
obtained as follows.
> summary(gol.rp)
Call:
8.3. CLASSIFICATION TREES 155
rpart(formula = gol.fac ~ golub[1042, ], method = "class")
n= 38
CP nsplit rel error xerror xstd
1 0.7272727 0 1.0000000 1.0000000 0.2541521
2 0.0100000 1 0.2727273 0.5454545 0.2043460
Node number 1: 38 observations, complexity param=0.7272727
predicted class=ALL expected loss=0.2894737
class counts: 27 11
probabilities: 0.711 0.289
left son=2 (26 obs) right son=3 (12 obs)
Primary splits:
golub[1042, ] < 1.198515 to the right, improve=10.37517, (0 missing)
Node number 2: 26 observations
predicted class=ALL expected loss=0.03846154
class counts: 25 1
probabilities: 0.962 0.038
Node number 3: 12 observations
predicted class=AML expected loss=0.1666667
class counts: 2 10
probabilities: 0.167 0.833
26
[1] 0.03846154
The expected loss in prediction accuracy of Node number 2 is 1/26 and that
of Node number 3 is 2/12. This equals the probabilities from the class counts.
The primary splits gives the estimated threshold value. To predict the class
of the individual patients one may use the function predict, as follows.
> predict(gol.rp,type="class")
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL AML ALL ALL ALL
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
AML ALL ALL ALL ALL ALL ALL AML ALL AML AML AML AML AML AML AML AML AML
Levels: ALL AML
156 CHAPTER 8. CLASSIFICATION METHODS
Hence, Patient 17 and 21 are erroneously predicted as AML and Patient 29
is erroneously predicted in the ALL class. A more precise output is obtained
by asking for the probability of class membership.
> predict(gol.rp, type="prob")
ALL AML
1 0.9615385 0.03846154
2 0.9615385 0.03846154
etc.
Based on this the probability of patient 21 to have ALL is 0.16 and that to
have AML is 0.83.
ALL AML

0
.
5
0
.
0
0
.
5
1
.
0
1
.
5
2
.
0
2
.
5
Figure 8.7: Boxplot of expression
values from gene CCND3 Cyclin
D3 for ALL and AML patients
golub[1042, ]>=1.199
ALL
25/1
AML
2/10
Figure 8.8: Classiﬁcation tree
of expression values from gene
CCND3 Cyclin D3 for classiﬁca-
tion of ALL and AML patients.
Example 4. Gene selection of the Golub (1999) data. By recursive
partitioning it is possible to select among the genes of Golub et al. (1999)
those which give the best partitioning. For the latter to work we have to
specify the gene expressions as the variables (columns). For this we use the
transposition operator t. To facilitate reading the output we add gene 1 to
gene 3051 as column names.
8.3. CLASSIFICATION TREES 157
library(rpart);data(golub); library(multtest)
row.names(golub)<- paste("gene", 1:3051, sep = "")
goldata <- data.frame(t(golub[1:3051,]))
gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
gol.rp <- rpart(gol.fac~., data=goldata, method="class", cp=0.001)
plot(gol.rp, branch=0,margin=0.1); text(gol.rp, digits=3, use.n=TRUE)
golub.gnames[896,]
Inspection of the plot yields gene ”FAH Fumarylacetoacetate” as the predic-
tor by which the two classes of patients can be predicted perfectly.
In order to further illustrate possibilities of classiﬁcation methods we use
the ALL data collected by Chiaretti, et al. (2004), see also Chapter 6.
Example 5. Application to the Chiaretti (2004) data. With respect to
the ALL data we want to predict from the gene expressions the diagnosis of B-
cell State B1, B2, and B3. Since the complete set of 12625 gene expressions is
too large, we select the genes with diﬀerent means over the patients groups. It
is obvious that only these gene can contribute to the prediction of the disease
states. In particular we select the gene with ANOVA p-value is smaller than
0.000001.
library("hgu95av2.db");library(ALL);data(ALL)
ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")] pano <- apply(exprs(ALLB123), 1, function(x) anova(lm(x ~ ALLB123$BT))$Pr[1]) names <- featureNames(ALL)[pano<0.000001] symb <- mget(names, env = hgu95av2SYMBOL) ALLBTnames <- ALLB123[names, ] probedat <- as.matrix(exprs(ALLBTnames)) row.names(probedat)<-unlist(symb) The probe symbols are extracted from the hgu95av2SYMBOL environment and used as row names to facilitate readability of the resulting tree. There are 78 patients selected and 29 probes. The recursive partitioning to ﬁnd the tree can be performed by the following script. > diagnosed <- factor(ALLBTnames$BT)
> tr <- rpart(factor(ALLBTnames$BT) ~ ., data = data.frame(t(probedat))) > plot(tr, branch=0,margin=0.1); text(tr, digits=3, use.n=TRUE) 158 CHAPTER 8. CLASSIFICATION METHODS > rpartpred <- predict(tr, type="class") > table(rpartpred,diagnosed) diagnosed rpartpred B1 B2 B3 B1 17 2 0 B2 1 33 5 B3 1 1 18 The rows to the left of the table give the frequencies of the predicted B cell stages and the columns on top the diagnosed B cell stages from the factor. The matrix with frequencies of the predicted and true patient status is often called a “confusion table”. The resulting tree in Figure 8.9 should be read as follows. If gene expression MME is strictly smaller than the cutoﬀ value 8.395, then the patient is predicted to be in state (class) B1. If the expression of LSM6 smaller than 4.192, then the predicted state is B2, and if it is larger than the predicted state it is B3. The misclassiﬁcation rate is 10/78=0.1282051, which is low, but not zero. It may happen that the probability of the predicted class is close to that of the diagnosed. An overview of the latter can be obtained as follows. predicted.class <- predict(tr, type="class") predicted.probabilities <- predict(tr, type="prob") out <- data.frame(predicted.probabilities,predicted.class, diagnosis=factor(ALLBTnames$BT))
> print(out,digits=2)
B1 B2 B3 predicted.class diagnosis
01005 0.026 0.85 0.13 B2 B2
01010 0.026 0.85 0.13 B2 B2
04006 0.895 0.11 0.00 B1 B1
04007 0.026 0.85 0.13 B2 B2
04008 0.895 0.11 0.00 B1 B1
04010 0.050 0.05 0.90 B3 B1
04016 0.895 0.11 0.00 B1 B1
06002 0.026 0.85 0.13 B2 B2
08001 0.026 0.85 0.13 B2 B2
08011 0.026 0.85 0.13 B2 B3
08012 0.026 0.85 0.13 B2 B3
08018 0.050 0.05 0.90 B3 B3
08024 0.895 0.11 0.00 B1 B2
8.3. CLASSIFICATION TREES 159
09008 0.026 0.85 0.13 B2 B3
...
For instance, the sixth patient is with probability .90 in class B3 and with
probability .05 in class B1, which is the diagnosed disease state.
MME< 8.395
LSM6< 4.192
B1
17/2/0
B2
1/33/5
B3
1/1/18
Figure 8.9: rpart on ALL B-cel 123
data.
40480_s_at
1173_g_at
37320_at
40729_s_at
36829_at
32716_at
32116_at
32977_at
34378_at
36711_at
37544_at
307_at
40440_at
38032_at
1389_at
0.35 0.45 0.55 0.65
MeanDecreaseAccuracy
32977_at
32716_at
307_at
34333_at
34347_at
35769_at
37043_at
37544_at
34891_at
36829_at
40493_at
40440_at
38032_at
36711_at
1389_at
0.0 0.2 0.4 0.6
MeanDecreaseGini
rf1
Figure 8.10: Variable importance
plot on ALL B-cell 123 data.
Note the reduction in variables from twenty nine to two in the actual
construction of the tree. In a construction like this the gene expressions
(variables) are linearly dependent in the sense that once the ﬁrst gene is
selected for the ﬁrst split, then highly similar ones are not selected anymore.
It can be instructive to leave out the variables selected from the data and to
redo the analysis.
A generally applied manner to evaluate an estimated model is by its pre-
dictive accuracy with respect to a future data set. When such a future data
set is not available, it is common practice to split the available data in two
parts: A training set and a validation set. Then the model is estimated from
the training set and this is used to predict the class of the patients in the
validation set. Then a confusion matrix is constructed with the frequencies
of true classes against predicted classes. Next, the misclassiﬁcation rate can
be computed to evaluate the predictive accuracy. This can very well be seen
160 CHAPTER 8. CLASSIFICATION METHODS
as a method to detect for over ﬁtting where the model estimates are so data
speciﬁc that generalization to future data sets is in danger.
Example 6. Training and validation. In the setting of B-cell ALL data
with State 1, 2, and 3 the manner to split the data centers around randomly
splitting the patients in two halves. The 78 patients in State 1, 2 or 3 can
be split in two halves, as follows.
i <- sample(1:78, 39, replace = FALSE)
noti <- setdiff(1:78,i)
df <- data.frame(Y = factor(ALLBTnames$BT), X =t(probedat)) rpart.est <- rpart(Y ~ ., data = df, subset=i) rpart.pred.t <- predict(rpart.est, df[i,], type="class") > table(rpart.pred.t,factor(ALLBTnames$BT[i]))
rpart.pred.t B1 B2 B3
B1 11 1 0
B2 0 12 0
B3 0 1 14
> rpart.pred.v <- predict(rpart.est,df[noti,], type="class")
> table(rpart.pred.v,factor(ALLBTnames$BT[noti])) rpart.pred.v B1 B2 B3 B1 6 1 0 B2 1 19 3 B3 1 2 6 The misclassiﬁcation rate in the training set is 2/39 = 0.05 and in the val- idation set is 7/39 = 0.18. Note that the diﬀerences mainly occur between State 2 and 3. Generally the prediction of disease state from the training set is better because the model is estimated from these data. The same split of the data into training and validation set will be used for other methods as well. 8.4 Support Vector Machine A support vector machine ﬁnds separating lines (hyper planes) between groups of points. This works like a classiﬁcation problem where the classes 8.4. SUPPORT VECTOR MACHINE 161 of patients are to be predicted from gene expression values. If such sepa- rating lines do exist in the data, then a linear support vector machine will ﬁnd these. This is because the optimization method behind it is based on quadratic programming by iterative algorithms which ﬁnd the globally opti- mal solution with certainty. Support vector machines do not automatically select variables and are designed for continuous predictor variables. Since the mathematical details are beyond the current scope, we shall conﬁne with illustrating applications to gene expression data. Example 1. Application to the Chiaretti (2004) data. The parameters for the support vector machine can be determined by the function svm from the e1071 package, as follows. library(e1071) df <- data.frame(Y = factor(ALLBTnames$BT), X =t(probedat))
Y <- factor(ALLBTnames$BT);X <- t(probedat) svmest <- svm(X, Y, data=df, type = "C-classification", kernel = "linear") svmpred <- predict(svmest, X, probability=TRUE) > table(svmpred, factor(ALLBTnames$BT))
svmpred B1 B2 B3
B1 19 0 0
B2 0 36 1
B3 0 0 22
The confusion matrix shows that the misclassiﬁcation rate of the three classes
of B-cell ALL is 1/78=0.0128 is very small, so that the prediction is almost
perfect. Note, however, from summary(svmest) that the number of support
vectors per class equals 20, 9, and 11, for class B1, B2, and B3, respectively.
These have values for all input variables (genes) as can be obtained from
dim(svmest$SV) and the coeﬃcient vectors dim(svmest$coefs). Hence,
the excellent prediction properties are obtained by a very large number of
estimated parameters.
Example 2. Training and validation. A generally applied manner to
evaluate the predictive quality of an estimated model is by splitting the data
into a training and a validation set. The model is estimated by the training
set and then the class of the patients in the validation set is predicted. We
shall use the same split as in Example 6 of the previous section.
162 CHAPTER 8. CLASSIFICATION METHODS
> Yt <- factor(ALLBTnames$BT)[i]; Yv <- factor(ALLBTnames$BT)[noti]
> X <- t(probedat); Xt <- X[i,]; Xv <- X[noti,]
> svmest <- svm(Xt, Yt, type = "C-classification", kernel = "linear")
> svmpredt <- predict(svmest, Xt, probability=TRUE)
> table(svmpredt, Yt)
Yt
svmpredt B1 B2 B3
B1 11 0 0
B2 0 14 0
B3 0 0 14
> svmpredv <- predict(svmest, Xv, probability=TRUE)
> table(svmpredv, Yv)
Yv
svmpredv B1 B2 B3
B1 5 0 0
B2 1 19 4
B3 2 3 5
The predictions of the disease states of the patients from the training set per-
fectly match the diagnosed states. The predictions, however, of the classes
of the patients from the validation set have misclassiﬁcation rate 10/39=0.25
and are therefore less accurate. Hence, the parameter estimates from the
training set are sample speciﬁc and do not generalize with the same accuracy
to the validation set.
8.5 Neural Networks
Neural networks are nonlinear models consisting of nonlinear hyperplanes
around classes of objects given a set of prediction variables (Ripley, 1996).
We conﬁne with illustrating the method by two examples.
Example 1. Application to the Chiaretti (2004) data. The models can
be estimated by the function nnet from the package that goes under the
same name. To avoid having to many variables we randomly select a subset
of 20 genes.
> Y <- factor(ALLBTnames$BT);X <- t(probedat) 8.5. NEURAL NETWORKS 163 > library(nnet) > df <- data.frame(Y = Y, X = X[, sample(ncol(X), 20)]) > nnest <- nnet(Y ~ .,data = df, size = 5, maxit = 500, decay = 0.01, + MaxNWts = 5000) > pred <- predict(nnest, type = "class") > table(pred, Y) # prints confusion ma Y pred B1 B2 B3 B1 19 0 0 B2 0 36 0 B3 0 0 23 The confusion matrix shows that zero out of 78 patients are mis-classiﬁed. Example 2. Training and validation. The results from cross validation on the neural networks are as follows. > nnest.t <- nnet(Y ~ ., data = df,subset=i, size = 5,decay = 0.01, + maxit=500) > prednnt <- predict(nnest.t, df[i,], type = "class") > table(prednnt,Ytrain=Y[i]) Ytrain prednnt B1 B2 B3 B1 11 0 0 B2 0 14 0 B3 0 0 14 > prednnv <- predict(nnest.t, df[noti,], type = "class") > table(prednnv, Yval= Y[noti]) Yval prednnv B1 B2 B3 B1 4 1 0 B2 4 17 4 B3 0 4 5 The predictions on the training set have misclassiﬁcation rate zero and that on the validation set 13/39=0.33. 164 CHAPTER 8. CLASSIFICATION METHODS 8.6 Generalized Linear Models Within the framework of generalized linear models the diagnosis of a patient is seen as a response. In case the response has the values healthy or disease for which it may be assumed that the binomial distribution holds with a succes probability p i . Recall from Chapter 3 that for a binomially distributed variable with y i successes out of n i it holds that the probability of y i successes out of ni equals P(Y i = y i ) = n i ! y i !(n i −y i )! p y i i (1 −p i ) n i −y i , for k = 0, · · · , n i . The value of p i is closely related to one or more predictor variables x 1 and x 2 via a linear combination. That is, the linear model holds such that η i = β 0 1 x i1 2 x i2 . The predictors are linked to the succes probability via the so-called logit link p i = e η i e η i + 1 = exp(β 0 + β 1 x i1 + β 2 x i2 ) 1 + exp(β 0 + β 1 x i1 + β 2 x i2 ) . Rather than going deeper into the details, the usefulness of generalized linear models will be illustrated with two examples. Example 1. CCND3 Cyclin D3. In the Golub et al. (1999) data we may model Y i = 1 if the patient is diagnosed as ALL and Y i = 0 if (s)he is diagnosed as AML. We use the CCND3 Cyclin D3 gene expression values as predictor. To will be convenient to compute the response by -golub.cl + 1. This yield 1 for ALL and 0 for not ALL 5 . library(faraway) logitmod <- glm((-golub.cl + 1) ~ golub[1042,], family=binomial(link = "logit")) pchisq(deviance(logitmod),df.residual(logitmod),lower=FALSE) plot((-golub.cl + 1) ~ golub[1042,], xlim=c(-2,5), ylim = c(0,1), xlab="CCND3 expression values ", ylab="Probability of ALL") x <- seq(-2,5,.1) lines(x,ilogit(-4.844124 + 4.439953*x)) pchisq(deviance(logitmod),df.residual(logitmod),lower=FALSE) 5 One may also conveniently use a factor as response variable 8.6. GENERALIZED LINEAR MODELS 165 > summary(logitmod) Call: glm(formula = (-golub.cl + 1) ~ golub[1042, ], family = binomial(link = "logit")) Coefficients: Estimate Std. Error z value Pr(>|z|) (Intercept) -4.844 1.849 -2.620 0.00880 ** golub[1042, ] 4.440 1.488 2.984 0.00284 ** Null deviance: 45.728 on 37 degrees of freedom Residual deviance: 18.270 on 36 degrees of freedom AIC: 22.270 From Figure 8.11 it can be seen that the logit curve ﬁts the data fairly well. From the summary of the output it can be seen that the estimated intercept is -4.844 and the estimated slope is 4.440. Both coeﬃcients are signiﬁcantly diﬀerent from zero. The goodness-of-ﬁt value of the model is computed from the chi-square distribution and equals .99. The model ﬁts the data well. The predictive accuracy of the model may be obtained as follows. > pred <- predict(logitmod,type="response") > 0.5 > pred.fac <- factor(pred,levels=c(TRUE,FALSE),labels=c("ALL","not ALL")) > table(pred.fac,gol.fac) gol.fac pred.fac ALL AML ALL 26 2 not ALL 1 9 The diagnosis of the majority of patients is predicted correctly. Example 2. Application to the Chiaretti (2004) data. With respect to the ALL data we want model the diagnosis of B-cell State B1, B2, and B3 as a response. The factor representing these levels can be used as input for the response. Here we use the gene expressions with greatest importance from the classiﬁcation tree in Section 8.3. We assign the biological name to the predictor variables and estimate the generalized linear model. library(nnet);library("hgu95av2.db");library(ALL);data(ALL) 166 CHAPTER 8. CLASSIFICATION METHODS probe.names <- c("1389_at","35991_at","40440_at") ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")]
probedat <- exprs(ALLB123)[probe.names,]
row.names(probedat) <- unlist(mget(probe.names, env = hgu95av2SYMBOL))
fac <- factor(ALLB123$BT,levels=c("B1","B2","B3")) dat <- data.frame(fac,t(probedat)) mnmod <- multinom(fac ~ ., family=binomial(link = "logit"),data=dat) > summary(mnmod) Call: multinom(formula = fac ~ ., data = dat, family = binomial(link = "logit")) Coefficients: (Intercept) MME LSM6 SERBP1 B2 14.36158 4.14002 -0.8494635 -5.104337 B3 -12.90584 4.94908 4.7415802 -5.655420 Std. Errors: (Intercept) MME LSM6 SERBP1 B2 16.36959 1.367058 1.716513 2.222486 B3 17.97896 1.424526 1.744425 2.313700 Residual Deviance: 59.88298 AIC: 75.88298 Apart from the intercepts, the estimated coeﬃcients are signiﬁcantly diﬀerent from zero. > predmn <- predict(mnmod,type="class") > table(predmn,fac) fac predmn B1 B2 B3 B1 17 2 1 B2 1 31 5 B3 1 3 17 The model predict the diagnosed classes quite well. Generalized linear models are statistical models which have to be esti- mated by an iterative process which may need some computation time. It 8.7. OVERVIEW AND CONCLUDING REMARKS 167 has the advantage that conﬁdence intervals or the signiﬁcance of the param- eters can be estimated. On the other hand, using statistical model building procedures may not designed to handle large amounts of predictor variables. Hence, the researcher does need to have some idea on which gene expressions (s)he want to use as predictors. Indeed, better models than those estimated in the above example may certainly exist. 8.7 Overview and concluding remarks Central themes in prediction methods are the face validity (clarity) of the model, the size of the model, and predictive accuracy on a validation set. For many researchers it is of crucial importance to have a clear idea on what a method is essentially doing. Some models and their estimation procedures are mathematically intricate and seem to be recollected in the mind of many researchers as black boxes. Even from a more pragmatic point of view such need not be devastating if the predictive accuracy is excellent. However, support vector machines and neural networks typically use a large number of parameters to predict well on a test set, but less well on validation sets. It is, furthermore, questionable whether a zero misclassiﬁcation rate is ra- tional since patients may be misclassiﬁed by the diagnosis or very close to transferring from one state to the other. Recursive partitioning to estimate a classiﬁcation tree performs very well on variable selection and pruning in order to discover as few variables (gene expressions) as possible for maximum predictive accuracy. In addition, it seems obvious that classiﬁcation trees have great clarity, see e.g. the CART package (Breiman et al., 1984) for further types of recursive trees. Note that several methods have diﬀerent misclassiﬁcation rates with respect to the whole sample, but comparable rates on the validation sets. It should, however, be clear that when there are non-linear relationships between pre- dictor variables and classes, then nonlinear models should outperform linear ones 6 . 8.8 Exercises 1. Classiﬁcation tree of Golub data. Use recursive partitioning in rpart 6 Some people may want to use the ade4TkGUI() 168 CHAPTER 8. CLASSIFICATION METHODS (a) Find a manner to identify an optimal gene with respect the Golub data to prediction of the ALL AML patients. (b) Explain what the code does. (c) Use rpart to construct the classiﬁcation tree with the genes that you found. Does it have perfect predictions? (d) Find the row number of gene Gdf5, which is supposed not to have any relationship with leukemia. Estimate a classiﬁcation tree and report the probability of misclassiﬁcation. Give explanations of the results. 2. Sensitivity versus speciﬁcity. (a) Produce a sensitivity versus speciﬁcity plot for the gene expression values of CCND3 Cyclin D3. (b) In what sense does it resemble Figure 8.2. (c) Compute the area under the curve for sensitivity versus speciﬁcity curve. 3. Comparing Classiﬁcation Methods. To obtain an idea on the misclas- siﬁcation rate when there is no relation between the predictors and the factor indicating groups, we perform a small simulation study. (a) Construct a factor with 100 values one and two and a matrix with predictor variables of 500 by 4 with values from the normal distribution. Use the ﬁrst four letters of the alphabet for the column names. (b) Use rpart to construct a recursive tree and report the misclassi- ﬁcation rate. Comment on the results. (c) Do the same for support vector machines. (d) Do the same for neural networks. (e) Think through your results and comment on these. 4. Prediction of achieved remission. For the ALL data from its ALL library the patients are checked for achieving remission. The variable ALL$CR
has values CR (became healthy) and REF (did not respond to therapy;
remain ill).
8.8. EXERCISES 169
(a) Construct an expression set containing the patients with values
on the phenotypical variable remission and the gene expressions
with a signiﬁcant p-value on the t-test with the patient groups CR
or REF.
(b) Use recursive partitioning to predict the remission. Report the
misclassiﬁcation rate and the names of the genes that play a role
in the tree.
5. Gene selection by area under the curve. A strategy of selecting genes
is to compute the auc for each gene and to use the best 10 for further
investigation. Compute the auc for each row with gene expressions of
the Golub at al. (1999) data. Collect these in a vector and select the
ten best. Is ”CCND3 Cyclin D3” among these?
6. Classiﬁcation Tree for Ecoli. The ecoli data can be download by the
following: (Hint: Copy two separated lines into one before running it.)
"http://www.grappa.univ-lille3.fr/~torre/Recherche/Datasets/
colnames(ecoli) <- c("SequenceName","mcg","gvh","lip","chg",
"aac","alm1","alm2","ecclass")
(a) Use ecclass to construct a factor containing the ”cp”,”im”,and
”pp”.
(b) Construct a classiﬁcation tree using the variables ”mcg”,”gvh”,”lip”,”aac”,”alm1”,”alm2”.
Give the code. Hint: Use the addition notation.
(c) Plot the tree and report the variables that play a role in the con-
structed tree.
(d) Predict the class by the tree. Report the code and the miss-
classiﬁcation rate.
(e) Leaf out the upper variable in the classiﬁcation tree and re-estimate
the tree. Report the miss-classiﬁcation rate. Is it much worse?
170 CHAPTER 8. CLASSIFICATION METHODS
Table 8.2: Ordered expression values of gene CCND3 Cyclin D3, index 2
indicates ALL, 1 indicates AML, cutoﬀ points, number of false positives,
false positive rate, number of true positives, true positive rate.
data index cutoﬀ fp fpr tp tpr
1 Inf 0 0.00 0 0.00
2 2.77 2 2.77 0 0.00 1 0.04
3 2.59 2 2.59 0 0.00 2 0.07
4 2.45 2 2.45 0 0.00 3 0.11
.
.
.
22 1.78 2 1.78 0 0.00 21 0.78
23 1.52 2 1.52 0 0.00 22 0.81
24 1.37 2 1.45 1 0.09 22 0.81
25 1.33 2 1.37 1 0.09 23 0.85
26 1.28 2 1.33 1 0.09 24 0.89
27 1.11 2 1.28 1 0.09 25 0.93
28 0.46 2 1.12 2 0.18 25 0.93
29 1.45 1 1.11 2 0.18 26 0.96
30 1.12 1 1.02 3 0.27 26 0.96
31 1.02 1 0.89 4 0.36 26 0.96
32 0.89 1 0.83 5 0.45 26 0.96
33 0.83 1 0.74 6 0.55 26 0.96
34 0.74 1 0.64 7 0.64 26 0.96
35 0.64 1 0.49 8 0.73 26 0.96
36 0.49 1 0.46 8 0.73 27 1.00
37 0.43 1 0.43 9 0.82 27 1.00
38 0.13 1 0.13 10 0.91 27 1.00
39 −0.74 1 −0.74 11 1.00 27 1.00
8.8. EXERCISES 171
−2 −1 0 1 2 3 4 5
0
.
0
0
.
2
0
.
4
0
.
6
0
.
8
1
.
0
CCND3 expression values
P
r
o
b
a
b
i
l
i
t
y

o
f

A
L
L
Figure 8.11: Logit ﬁt to the CCND3 Cyclin D3 expression values.
172 CHAPTER 8. CLASSIFICATION METHODS
Chapter 9
Analyzing Sequences
For many purposes in bioinformatics nucleotide or amino acid sequences are
analyzed. The idea is that highly similar sequences may have identical bi-
ological functions. For expressing the similarity of sequences it is necessary
to compute ﬁrst their optimal alignment. It will be explained and illustrated
how optimal pairwise alignment can be obtained. Furthermore, it is of im-
portance to compute quantities for DNA sequences such as the CG fraction,
or, for amino acid sequences, the isoelectric point or the hydropathy score.
It will be explained and illustrated how such quantities can be computed.
In this chapter you learn how to query online data bases, to translate RNA
into protein sequences, to match patterns, and to program pairwise align-
ments. We will start, however, with a query language in order to download
sequences.
9.1 Using a query language
It will be illustrated how the query language from the seqinr package can be
it is important to know which banks can be chosen.
> library(seqinr)
> choosebank()
[1] "genbank" "embl" "emblwgs" "swissprot" "ensembl"
[6] "refseq" "nrsub" "hobacnucl" "hobacprot" "hovergendna"
[11] "hovergen" "hogenom" "hogenomdna" "hogennucl" "hogenprot"
[16] "hoverclnu" "hoverclpr" "homolens" "homolensdna" "greview"
173
174 CHAPTER 9. ANALYZING SEQUENCES
[21] "polymorphix" "emglib" "HAMAPnucl" "HAMAPprot" "hoppsigen"
[26] "nurebnucl" "nurebprot" "taxobacgen"
There are many possibilities to use the query language e.g. for answering
questions about sequences from online data bases (Gouy, et al. 1984). We
give a few examples to illustrate some of its possibilities. For this we shall
temporary use the option virtual=TRUE to save time by preventing actual
1
We may ask: How many ccnd sequences has genbank?
> choosebank("genbank")
> query("ccnd","k=ccnd",virtual=TRUE)$nelem [1] 147 More speciﬁc: How many sequences ccnd sequences has genbank for the species homo sapiens. > query("ccnd3hs","sp=homo sapiens AND k=ccnd3",virtual=TRUE)$nelem
[1] 9
For many other combinations of search options we refer to the manual of
the seqinr package and for a book length treatment with many examples to
Charif et al. (2008).
quences
After sequences are downloaded in binary format it is essential to obtain
information with respect to their accession number, length, actual elements,
translation to amino acids, and annotation. How to do this will brieﬂy be
illustrated by an example.
ens and a gene name like ”CCND3”.
> choosebank("genbank")
> query("ccnd3hs","sp=homo sapiens AND k=ccnd3@")
> ccnd3hs$nelem [1] 9 1 The results below are obviously time dependent. 9.2. GETTING INFORMATION ON DOWNLOADED SEQUENCES 175 The sequences are downloaded in binary format. The symbol @ acts as a wildcard for any zero or other characters. There are a number of useful functions available to obtain further information. Some of these are getName, getLength, getSequence, getTrans, and getAnnot. To use these on a list containing sets of sequences the functionality sapply is very convenient. This is illustrated by extracting the NCBI accession numbers. > sapply(ccnd3hs$req, getName)
[1] "AF517525.CCND3" "AL160163.CCND3" "AL160163.PE5" "AL161651"
[5] "BC011616.CCND3" "CR542246" "HUMCCND3A.CCND3" "HUMCCND3PS.PE1"
[9] "HUMCCNDB04.CCND3" "HUMCYCD3A.CCND3"
The length of the sequences can be obtained by the getLength function.
> sapply(ccnd3hs$req, getLength) [1] "879" "879" "729" "211627" "879" "879" "879" "537" "559" "879" Let’s obtain the ﬁrst sequence and print its ﬁrst ﬁfteen nucleotides to the screen. 2 > getSequence(ccnd3hs$req[[1]])[1:15]
[1] "a" "t" "g" "g" "a" "g" "c" "t" "g" "c" "t" "g" "t" "g" "t"
Its translation into amino acids can be obtained
> getTrans(ccnd3hs$req[[1]])[1:15] [1] "M" "E" "L" "L" "C" "C" "E" "G" "T" "R" "H" "A" "P" "R" "A" as well as its annotation from the corresponding web page: > getAnnot(ccnd3hs$req[[1]])
[1] " CDS join(1051..1248,2115..2330,5306..5465,6005..6141,"
[2] " 6593..6760)"
[3] " /gene=\"CCND3\""
[4] " /codon_start=1"
[5] " /product=\"cyclin D3\""
[6] " /protein_id=\"AAM51826.1\""
[7] " /db_xref=\"GI:21397158\""
[8] " /translation=\"MELLCCEGTRHAPRAGPDPRLLGDQRVLQSLLRLEERYVPRASY"
2
Use double brackets to extract a sequence from a list.
176 CHAPTER 9. ANALYZING SEQUENCES
[9] " FQCVQREIKPHMRKMLAYWMLEVCEEQRCEEEVFPLAMNYLDRYLSCVPTRKAQLQLL"
[11] " LAFILHRLSLPRDRQALVKKHAQTFLALCATDYTFAMYPPSMIATGSIGAAVQGLGAC"
[12] " SMSGDELTELLAGITGTEVDCLRACQEQIEAALRESLREAAQTSSSPAPKAPRGSSSQ"
[13] " GPSQTSTPTDVTAIHL\""
9.3 Computations on sequences
A basic quantity to compute are the nucleotide and the dinucleotide frequen-
cies.
Example 1. Frequencies of (di)nucleotides. We shall continue with
the ﬁrst result from the CCND3 (Cyclin D3) search with accession num-
ber ”AF517525.CCND3”. To compute the frequencies we may extract the
sequence from a list in order to use the basic function table, as follows.
> table(getSequence(ccnd3hs$req[[1]])) a c g t 162 288 267 162 This table can also be computed by the seqinr function count, which is more general in the sense that frequencies of dinucleotides can be computed. > count(getSequence(ccnd3hs$req[[1]]),2)
aa ac ag at ca cc cg ct ga gc gg gt ta tc tg tt
25 44 64 29 68 97 45 78 52 104 76 34 16 43 82 21
This will be quite useful in the next chapter. Indeed, changing 2 into 3 makes
it possible to count trinucleotides.
Example 2. G + C percentage. We are often interested in the fraction
G plus C in general (GC), or starting from the ﬁrst position of the codon
bases (GC1), the second (GC2), or third (GC3).
> GC(getSequence(ccnd3hs$req[[1]])) [1] 0.6313993 9.3. COMPUTATIONS ON SEQUENCES 177 > GC1(getSequence(ccnd3hs$req[[1]]))
[1] 0.6484642
> GC2(getSequence(ccnd3hs$req[[1]])) [1] 0.4641638 > GC3(getSequence(ccnd3hs$req[[1]]))
[1] 0.78157
Hence, the G + C percentage is largest when started at position three. It
is also possible to compute the G + C fraction in a window of length 50 nt,
say, and to plot it along the sequence.
GCperc <- double()
n <- length(ccnd3[[1]])
for (i in 1:(n - 50)) GCperc[i] <- GC(ccnd3[[1]][i:(i+50)])
plot(GCperc,type="l")
By double() we ﬁrst create a vector. From Figure 9.1 it can be seen that
the G + C fraction changes drastically along a window of 50 nucleotides.
With respect to over or under representation of dinucleotides there is a func-
tion ρ (rho) available, which is deﬁned as
ρ(xy) =
f
xy
f
x
· f
y
,
where f
xy
, f
x
, and f
y
are the frequencies of the (di)nucleotide xy, x, and y,
respectively. The z-score is computed by subtracting the mean and dividing
by the standard deviation (Palmeira, et al., 2006). The latter is somewhat
more sensitive for over and under representation.
Example 3. Rho and z-scores. The coeﬃcient rho and the corresponding
z-scores will be computed from the sequence with NCBI accession number
”AF517525.CCND3”.
> round(rho(getSequence(ccnd3hs$req[[1]])),2) aa ac ag at ca cc cg ct ga gc gg gt ta tc tg tt 0.84 0.83 1.30 0.97 1.28 1.03 0.51 1.47 1.06 1.19 0.94 0.69 0.54 0.81 1.67 0.70 > round(zscore(getSequence(ccnd3hs$req[[1]]),modele=’base’),2)
178 CHAPTER 9. ANALYZING SEQUENCES
0 200 400 600 800
0
.
5
0
.
6
0
.
7
0
.
8
0
.
9
Index
G
C
p
e
r
c
Figure 9.1: G + C fraction of sequence ”AF517525.CCND3” along a window
of length 50 nt.
aa ac ag at ca cc cg ct ga gc gg gt ta
-1.08 -1.67 2.81 -0.18 2.78 0.42 -6.63 4.64 0.54 2.60 -0.80 -2.87 -3.10
tc tg tt
-1.86 6.22 -1.98
The rho value for CG is not extreme, but its z-score certainly is.
In case we have an amino acid sequence it may be useful to obtain a
plot of the amino acid frequencies. When we have translated the nucleotide
sequence into an amino acid sequence, it may be interesting to construct a
9.3. COMPUTATIONS ON SEQUENCES 179
plot expressing their frequencies. Such can be useful for a ﬁrst impression on
sequence similarity.
Example 4. Comparing Amino acid frequencies. We continue with the
ﬁrst result from the CCND3 (Cyclin D3) search, translate and order it, and,
next, produce a dotchart with amino acid frequencies.
tab <- table(getTrans(ccnd3hs$req[[1]])) taborder <- tab[order(tab)] names(taborder) <- aaa(names(taborder)) dotchart(taborder,pch=19,xlab="Stop and amino-acid-counts") abline(v=1,lty=2) The script was run on both sequences AF517525.CCND3 and AL160163.CCND3 resulting in Figure 9.2 and 9.3, respectively. The two sequences are highly similar with respect to amino acid frequencies. Stp Asn Trp Phe His Tyr Met Ile Lys Cys Asp Gly Val Gln Pro Thr Ser Glu Arg Ala Leu 0 10 20 30 40 Stop and amino−acid−counts Figure 9.2: Frequency plot of amino acids from accession num- ber AF517525.CCND3. Stp Asn Trp Phe His Tyr Met Ile Lys Cys Asp Gly Val Gln Pro Thr Ser Glu Arg Ala Leu 0 10 20 30 40 Stop and amino−acid−counts Figure 9.3: Frequency plot of amino acids from accession num- ber AL160163.CCND3. For amino acid sequences it may be of importance to compute the the- oretical isoelectric point or the molecular weight of the corresponding protein. 180 CHAPTER 9. ANALYZING SEQUENCES Example 5. Isoelectric point. The function computePI computes the theoretical isoelectric point of a protein, which is the pH at which the protein has a neutral charge (Gasteiger, et al. 2005). > computePI(getTrans(ccnd3hs$req[[1]]))
[1] 6.657579
The protein molecular weight can be computed as follows.
> pmw(getTrans(getSequence(ccnd3hs$req[[1]]))) [1] 32503.38 Note that it is easy to compute these for all downloaded proteins and to compare these. Another important quantity is hydropathy score (Kyte & Doolittle, 1982) of proteins, which is deﬁned as a weighted sum 20 i=1 α i f i of amino acid co- eﬃcients α i and the relative frequencies f i . An example will illustrate how it can be computed. Example 6. Hydropathy score. The coeﬃcients α 1 , · · · , α 20 are available as KD data from the EXP list of the seqinr package. The unique names are lexicographically ordered and stored in the object kdc. The scale is changed by the minus sign below so that hydrophilic proteins are positive, but smaller than one. A function is deﬁned to compute the hydropathy score for a set of amino acid sequences. ccnd3 <- sapply(ccnd3hs$req, getSequence)
ccnd3transl <- sapply(ccnd3, getTrans)
data(EXP)
names(EXP$KD) <- sapply(words(), function(x) translate(s2c(x))) kdc <- EXP$KD[unique(names(EXP$KD))] kdc <- -kdc[order(names(kdc))] linform <- function(data, coef) { #data are sequences f <- function(x) { freq <- table(factor(x, levels = names(coef)))/length(x) return(coef %*% freq) } res <- sapply(data, f) names(res) <- NULL 9.4. MATCHING PATTERNS 181 return(res) } kdath <- linform(ccnd3transl, kdc) > print(kdath,digits=3) [1] 0.0874 0.0962 0.0189 0.1496 0.0962 0.0874 0.0874 0.2659 0.2220 Indeed, the largest score is still much smaller than one, so the conclusion is that there are no hydrophilic proteins among our sequences. The data set aaindex of the seqinr library contains more than ﬁve hun- dred sets of coeﬃcients for computing speciﬁc quantities with respect to proteins. 9.4 Matching patterns A manner to investigate a long sequence is to search for identical patterns, eventually allowing for a speciﬁed number of mismatches. There are many relevant examples such as seeking for one of the stop codons UAG, UGA UAA in RNA, or recognition sequences of enzymes (e.g. Roberts, et al., 2007). We sustain with a brief example. Example 1. Pattern match. In the sequence with NCBI accession number ”AF517525.CCND3”, we seek the pattern ”cccggg” with zero mismatches as well as those with a single mismatch. By the function c2s a sequence of characters is converted into a single string. library(seqinr) choosebank("genbank") query("ccnd3hs","sp=homo sapiens AND k=ccnd3@") ccnd3 <- sapply(ccnd3hs$req, getSequence)
ccnd3nr1 <- c2s(ccnd3[[1]])
> ccnd3nr1
[1] "atggagctgctgtgttgcgaaggcacccggcacgcgccccgggccgggccggacccgcgg"...
> subseq <- "cccggg"
> countPattern(subseq, ccnd3nr1, mismatch = 0)
[1] 2
> matchPattern(subseq, ccnd3nr1, mismatch = 0)
Views on a 879-letter BString subject
182 CHAPTER 9. ANALYZING SEQUENCES
Subject: atggagctgctgtgttgcgaaggcacccggcacg...actcctacagatgtcacag
Views:
start end width
[1] 38 43 6 [cccggg]
[2] 809 814 6 [cccggg]
> matchPattern(subseq, ccnd3nr1, mismatch = 1)
Views on a 879-letter BString subject
Subject: atggagctgctgtgttgcgaaggcacccggcacg...actcctacagatgtcacag
Views:
start end width
[1] 26 31 6 [cccggc]
[2] 37 42 6 [ccccgg]
[3] 38 43 6 [cccggg]
[4] 43 48 6 [gccggg]
[5] 54 59 6 [cccgcg]
[6] 119 124 6 [cccgcg]
[7] 236 241 6 [ccctgg]
[8] 303 308 6 [cctggg]
[9] 512 517 6 [cccgtg]
[10] 612 617 6 [cacggg]
[11] 642 647 6 [cctggg]
[12] 661 666 6 [tccggg]
[13] 662 667 6 [ccgggg]
[14] 808 813 6 [ccccgg]
[15] 809 814 6 [cccggg]
[16] 810 815 6 [ccgggg]
The number of counted patterns allowing two mismatches is much larger.
9.5 Pairwise alignments
Among the basic questions about genes or proteins is to what extent a pair
of sequences are similar. To ﬁnd this out these are aligned in a certain man-
ner after which a similarity score can be computed. In order to understand
sequence alignment it is fundamental to have some idea about recursion.
9.5. PAIRWISE ALIGNMENTS 183
Example 1. Basic recursion. The idea of recursion is to generate a sequence
by deﬁning the current value as a function of the previous. Suppose that the
ﬁrst element is one, x
1
= 1, and that the sequence is deﬁned by
x
i
= x
i−1
+ 1.
Then we obtain x
1
= 1, x
2
= 2, x
3
= 3, etc, so that the sequence becomes
1, 2, 3, · · ·. Indeed, this is as fundamental as counting.
Another manner to deﬁne a sequence is by multiplying the previous value
by a constant. For example, let x
i
= 2x
i−1
with x
1
= 1. Then the values of
the sequence are x
1
= 1, x
2
= 2, x
3
= 4, x
3
= 8, etc. Also we see that in fact
x
n
= 2
n
, so that a value of the sequence can be computed without actually
computing all previous elements.
Another example would be x
i
= 2x
i−1
− 10, with x
1
= 1. In order to
compute the value x
10
we may use R, as follows.
> x<-double();x[1]<-1
> for (i in 2:10) {x[i]<- 2*x[i-1]-10}
> x[10]
[1] -4598
This illustrates basic ideas about recursively deﬁned sequences.
Suppose we want to compute an alignment score for two small DNA
sequences GAATTC and GATTA (Durbin et. al., 1998, p.18). We agree
that a match between two letters should have the score +2 and a mismatch
the score -1. A gap at a certain position of the sequences should be punished
by subtracting a score by d = 2. A possible alignment is
G
G
A
A
A
T
T
T
T

C
A
, where
the minus sign indicates a gap. Then the alignment consists of a match,
match, mismatch, match, gap, mismatch, respectively, so that the score is
2 + 2 − 1 + 2 − 2 − 1 = 2. Now the question is whether this alignment is
optimal in the sense that the score is maximal? The answer is: No! To see
this, consider the alignment
G
G
A
A
A

T
T
T
T
C
A
. Then we have a match, match, gap,
match, match, mismatch, respectively, so that the score is 2+2−2+2+2−1 =
5. This is better, but still we do not know whether this alignment is optimal.
In order to ascertain that the alignment is optimal we have to build an
alignment score matrix F(i, j). To do so it is convenient to start with building
the (mis)match score matrix s(i, j). Its (i, j)th element s(i, j) has the value
2 in case of a match and the value -1 is case of a mismatch. Note that for
184 CHAPTER 9. ANALYZING SEQUENCES
each step we can choose between a gap, a match, or a mismatch. Building
up the matrix F(i, j) recursively, means that we deﬁne its elements on the
basis of the values of its preceding elements. That is, given the values of
the previous elements F(i −1, j −1), F(i −1, j), and F(i, j −1), we will be
able to ﬁnd the best consecutive value for F(i, j). In particular, in case of
a match or a mismatch, we take F(i, j) = F(i − 1, j − 1) + s(x
i
, y
j
) and in
case of a gap we take F(i, j) = F(i − 1, j) − d or F(i, j) = F(i, j − 1) − d.
The famous Needleman-Wunsch alignment algorithm consists of taking the
maximum out of these possibilities at each step (e.g, Durbin et. al., 1998,
p.21). Their algorithm can be summarized, as follows.
F(i, j) = max
_
_
_
F(i −1, j −1) + s(i, j)
F(i −1, j) −d
F(i, j −1) −d
Note, however, that this will not yet work because we have not deﬁned any
initial values. In fact we will agree to start with F(0, 0) = 0 and due to the
gap penalties we take F(i, 0) = −id for the ﬁrst column and F(0, j) = −jd
for the ﬁrst row. Then, the ﬁnal score F(n, m) is the optimal score and the
values of the matrix F(i, j) indicates the optimal path. By informaticians
this recursive scheme is often called a “dynamic programming algorithm”.
Example 2. Dynamic programming of DNA sequences. Consider again
the DNA sequences GAATTC, GATTA, the score +2 for a match, -1 for a
mismatch, and the gap penalty d = 2. It is clarifying to ﬁrst construct the
score matrix s(i, j). For this we use the string-to-character function s2c, a
for loop, and an if else statement.
library(seqinr)
x <- s2c("GAATTC"); y <- s2c("GATTA"); d <- 2
s <- matrix(data=NA,nrow=length(y),ncol=length(x))
for (i in 1:(nrow(s))) for (j in 1:(ncol(s)))
{if (y[i]==x[j]) s[i,j]<- 2 else s[i,j]<- -1 }
rownames(s) <- c(y); colnames(s) <- c(x)
> s
G A A T T C
G 2 -1 -1 -1 -1 -1
A -1 2 2 -1 -1 -1
T -1 -1 -1 2 2 -1
9.5. PAIRWISE ALIGNMENTS 185
T -1 -1 -1 2 2 -1
A -1 2 2 -1 -1 -1
To initialize the ﬁrst row and column of the matrix F(i, j), it is convenient
to use the function seq. The purpose of the max function seems obvious.
F <- matrix(data=NA,nrow=(length(y)+1),ncol=(length(x)+1))
rownames(F) <- c("",y); colnames(F) <- c("",x)
F[,1] <- -seq(0,length(y)*d,d); F[1,] <- -seq(0,length(x)*d,d)
for (i in 2:(nrow(F)))
for (j in 2:(ncol(F)))
{F[i,j] <- max(c(F[i-1,j-1]+s[i-1,j-1],F[i-1,j]-d,F[i,j-1]-d))}
> F
G A A T T C
0 -2 -4 -6 -8 -10 -12
G -2 2 0 -2 -4 -6 -8
A -4 0 4 2 0 -2 -4
T -6 -2 2 3 4 2 0
T -8 -4 0 1 5 6 4
A -10 -6 -2 2 3 4 5
From the lower corner to the right hand side we see that the optimal score
is indeed 5.
Optimal alignment for pairs of amino acid sequences are often considered
to be more relevant because these are more closely related to biological func-
tions. For this purpose we may modify the previous scheme by changing the
gap penalty d and the (mis)match scores s(i, j). In particular, we shall use
the gap penalty d = 8 and for the (mis)match the scores from the so-called
BLOSUM50 matrix.
Example 3. Programming Needleman-Wunsch. For the two sequences
”PAWHEAE” and ”HEAGAWGHEE” (see, Durbin et. al., 1998, p.21) we
seek the Needleman-Wunsch optimal alignment score, using the BLOSUM50
(mis)match score matrix and gap penalty d = 8. You can either directly read
a BLOSUM matrix from NCBI
> file <- "ftp://ftp.ncbi.nih.gov/blast/matrices/BLOSUM50"
186 CHAPTER 9. ANALYZING SEQUENCES
Table 9.1: BLOSUM50 matrix.
A R N D C Q E G H I L K M F P S T W Y V
A 5 -2 -1 -2 -1 -1 -1 0 -2 -1 -2 -1 -1 -3 -1 1 0 -3 -2 0
R -2 7 -1 -2 -4 1 0 -3 0 -4 -3 3 -2 -3 -3 -1 -1 -3 -1 -3
N -1 -1 7 2 -2 0 0 0 1 -3 -4 0 -2 -4 -2 1 0 -4 -2 -3
D -2 -2 2 8 -4 0 2 -1 -1 -4 -4 -1 -4 -5 -1 0 -1 -5 -3 -4
C -1 -4 -2 -4 13 -3 -3 -3 -3 -2 -2 -3 -2 -2 -4 -1 -1 -5 -3 -1
Q -1 1 0 0 -3 7 2 -2 1 -3 -2 2 0 -4 -1 0 -1 -1 -1 -3
E -1 0 0 2 -3 2 6 -3 0 -4 -3 1 -2 -3 -1 -1 -1 -3 -2 -3
G 0 -3 0 -1 -3 -2 -3 8 -2 -4 -4 -2 -3 -4 -2 0 -2 -3 -3 -4
H -2 0 1 -1 -3 1 0 -2 10 -4 -3 0 -1 -1 -2 -1 -2 -3 2 -4
I -1 -4 -3 -4 -2 -3 -4 -4 -4 5 2 -3 2 0 -3 -3 -1 -3 -1 4
L -2 -3 -4 -4 -2 -2 -3 -4 -3 2 5 -3 3 1 -4 -3 -1 -2 -1 1
K -1 3 0 -1 -3 2 1 -2 0 -3 -3 6 -2 -4 -1 0 -1 -3 -2 -3
M -1 -2 -2 -4 -2 0 -2 -3 -1 2 3 -2 7 0 -3 -2 -1 -1 0 1
F -3 -3 -4 -5 -2 -4 -3 -4 -1 0 1 -4 0 8 -4 -3 -2 1 4 -1
P -1 -3 -2 -1 -4 -1 -1 -2 -2 -3 -4 -1 -3 -4 10 -1 -1 -4 -3 -3
S 1 -1 1 0 -1 0 -1 0 -1 -3 -3 0 -2 -3 -1 5 2 -4 -2 -2
T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 2 5 -3 -2 0
W -3 -3 -4 -5 -5 -1 -3 -3 -3 -3 -2 -3 -1 1 -4 -4 -3 15 2 -3
Y -2 -1 -2 -3 -3 -1 -2 -3 2 -1 -1 -2 0 4 -3 -2 -2 2 8 -1
V 0 -3 -3 -4 -1 -3 -3 -4 -4 4 1 -3 1 -1 -3 -2 0 -3 -1 5
or load a BLOSUM matrix from the Biostrings package. For the sake of
clarity we shall conveniently construct the matrix s(i, j) without any concern
library(seqinr);library(Biostrings);data(BLOSUM50)
x <- s2c("HEAGAWGHEE"); y <- s2c("PAWHEAE"); s <- BLOSUM50[y,x]; d <- 8
F <- matrix(data=NA,nrow=(length(y)+1),ncol=(length(x)+1))
F[1,] <- -seq(0,80,8); F[,1] <- -seq(0,56,8)
rownames(F) <- c("",y); colnames(F) <- c("",x)
for (i in 2:(nrow(F)))
for (j in 2:(ncol(F)))
{F[i,j] <- max(c(F[i-1,j-1]+s[i-1,j-1],F[i-1,j]-d,F[i,j-1]-d))}
9.5. PAIRWISE ALIGNMENTS 187
> F
H E A G A W G H E E
0 -8 -16 -24 -32 -40 -48 -56 -64 -72 -80
P -8 -2 -9 -17 -25 -33 -41 -49 -57 -65 -73
A -16 -10 -3 -4 -12 -20 -28 -36 -44 -52 -60
W -24 -18 -11 -6 -7 -15 -5 -13 -21 -29 -37
H -32 -14 -18 -13 -8 -9 -13 -7 -3 -11 -19
E -40 -22 -8 -16 -16 -9 -12 -15 -7 3 -5
A -48 -30 -16 -3 -11 -11 -12 -12 -15 -5 2
E -56 -38 -24 -11 -6 -12 -14 -15 -12 -9 1
Hence, from the lower-right corner we observe that the optimal score equals
one.
Example 4. Needleman-Wunsch. We may also conveniently use the pairwiseAlignment
function from the Biostrings package to ﬁnd the optimal Needleman-Wunsch
aligment score for the sequences PAWHEAE” and ”HEAGAWGHEE” (see,
Durbin et. al., 1998, p.21).
library(Biostrings);data(BLOSUM50)
> pairwiseAlignment(AAString("PAWHEAE"), AAString("HEAGAWGHEE"),
+ substitutionMatrix = "BLOSUM50",gapOpening = 0, gapExtension = -8,
+ scoreOnly = FALSE)
Global Pairwise Alignment
1: --P-AW-HEAE
2: HEAGAWGHE-E
Score: 1
Hence, we obtain the optimal score 1 as well as a representation of the opti-
mal alignment.
An obvious question is whether in the previous example the obtained
score 1 is to be evaluated as being “large” or not. A manner to answer this
question is by comparing it with the alignment score of random sequences.
That is, we may compute the probability of alignment scores larger than 1.
Example 5. Comparing with random sequences. To illustrate how the
probability of alignment scores larger than 1 can be computed we sample
randomly from the names of the amino acids, seven for y and 10 for x and
188 CHAPTER 9. ANALYZING SEQUENCES
compute the maximum alignment score. This is repeated 1000 times and the
probability of optimal alignment scores greater than 1 is estimated by the
corresponding proportion.
library(seqinr);library(Biostrings);data(BLOSUM50)
randallscore <- double()
for (i in 1:1000) {
x <- c2s(sample(rownames(BLOSUM50),7, replace=TRUE))
y <- c2s(sample(rownames(BLOSUM50),10, replace=TRUE))
randallscore[i] <- pairwiseAlignment(AAString(x), AAString(y),
substitutionMatrix = "BLOSUM50",gapOpening = 0, gapExtension = -8,
scoreOnly = TRUE)
}
> sum(randallscore>1)/1000
[1] 0.003
By the option scoreOnly = TRUE the optimal score is written to the vector
randallscore. The probability of scores larger than 1 equals 0.003 and is
therefore small and the alignment is stronger than expected from randomly
constructed sequences.
Example 6. Sliding window on Needleman-Wunsch scores. We may also
program a sliding window such that for each the Needleman-Wunsch align-
ment score is computed. Then the maximum can be found and localized.
choosebank("genbank"); library(seqinr)
query("ccnd3hs","sp=homo sapiens AND k=ccnd3@")
ccnd3 <- sapply(ccnd3hsreq, getSequence) ccnd3transl <- sapply(ccnd3, getTrans) x <- c2s(ccnd3transl[[1]]) y <- c2s(ccnd3transl[[1]][50:70]) nwscore <- double() ; n <- length(ccnd3transl[[1]]) for (i in 1:(n-21)) nwscore[i] <- pairwiseAlignment(AAString(c2s(ccnd3transl[[1]][i:(i+20)])), AAString(y),substitutionMatrix = "BLOSUM50",gapOpening = 0, gapExtension = -8, scoreOnly = TRUE) > pairwiseAlignment(AAString(y), AAString(y), substitutionMatrix = "BLOSUM50", gapOpening = 0, gapExtension = -8, 9.6. OVERVIEW AND CONCLUDING REMARKS 189 + scoreOnly = TRUE) [1] 152 > max(nwscore) [1] 152 > which.max(nwscore) [1] 50 Note that the maximum occurs when the subsequences are identical. The value of the maximum is 152 which occurs at position 50. 9.6 Overview and concluding remarks It was illustrated how the query language of the seqinr library can be used to download sequences, to translate these and to compute relevant quanti- ties such as the isoelectric point or the hydropathy score. Furthermore, it was illustated how patterns can be matched and how algorithms for optimal pairwise alignment can be programmed. Further applications are given by the exercises below. The package Biostrings contains the various PAM matrices for optimal alignment, as well as facilities to ﬁnd palindromes, and to read and write data in FASTA format (readFASTA). 9.7 Exercises 1. Writing to a FASTA ﬁle. Read, similar to the above, the ccnd3 se- quences using the query language and write the ﬁrst sequence to a ﬁles in FASTA format. Also try to write them all to FASTA format. 2. Dotplot of sequences. Use the function dotPlot of the seqinr package and par(mfrow=c(1,2)) to produce two adjacent plots. (a) Construct two random sequence of size 100 and plot the ﬁrst against second and the ﬁrst against the ﬁrst. (b) Construct a plot of the ﬁrst against the ﬁrst and the ﬁrst against the ﬁrst in reverse order. 190 CHAPTER 9. ANALYZING SEQUENCES (c) Download the sequences related to the species homo sapiens and a gene name like ”CCND3 Cyclin D3”. Construct a dotplot of the most similar and the least similar sequences. Report your observations. 3. Local alignment. The Smith-Waterman algorithm seeks maximum lo- cal alignment between subsequences of sequences. Their algorithm can be summarized (Durbin et al., 2005, p.22), as follows. F(i, j) = max _ _ _ F(i −1, j −1) + s(i, j) F(i −1, j) −d F(i, j −1) −d The algorithm allows the score zero if the others have negative values. The idea is that the maximum alignment can occur anywhere in the matrix, optimal alignment is deﬁnes as the maximum over the whole matrix. Program the Smith-Waterman algorithm and ﬁnd the optimal local alignment of the sequences PAWHEAE” and ”HEAGAWGHEE”. 4. Probability of more extreme alignment score. Sample x and y randomly from the names of the amino acids, seven for y and 10 for x. repeat this 1000 times and compute the optimal alignment score and use it to evaluate the signiﬁcance of the previously obtained score. 5. Prochlorococcus marinus. Each of three strains of P. marinus is ex- posed to diﬀerent intensities of UV radiation because these live in dif- ferent depths in water. The MIT 9313 strain lives at depth 135 m, SS120 at 120 m, and MED4 at 5 m. The latter strain is considered to be high-light-adapted. The residual intensities of 260-nm UVb ir- radiation corresponding to the given depths is 0.00007%, 0.0002% and 70%, respectively. It is hypothesized that the G + C content depends on the amount of radiation. The accession numbers of Gen bank are AE017126, BX548174, and BX548175, respectively. (a) Use the operator OR together with the accession numbers to download the sequences of the bacteria strains. (b) Compte the GC fraction of each of the sequences. (c) Is there a relation between UVb radiation and GC fraction? (d) Formulate a relevant hypothesis and test it. 9.7. EXERCISES 191 6. Sequence equality. Download the sequences ”AF517525.CCND3” and ”AL160163.CCND3”. Hint: These are the ﬁrst two from the query ”ccnd3” within homo sapiens. (a) Compute the length of the sequences. (b) Translate the sequences into amino acids and compare their fre- quencies. (c) Are they equal or, if not, in what position do they diﬀer? 7. Conserved region. At http://blocks.fhcrc.org there are blocks of highly conserved regions for proteins in PROSITE. Find PR00851A which contains blocks of protein related to a human gene responsible for DNA-repair defect xeroderma pigmentosum (sensitivity to ultravi- olet light) Perform a pairwise alignment with these subsequences and report the ones most and least similar. Use BLOSUM50. 8. Plot of CG proportion from Celegans. (a) Produce a plot of the CG proportion of the chromosome I of Cel- egans (Celegans.UCSC.ce2) along a window of 100 nucleotides. Take the ﬁrst 10,000 nucleotides. (b) A binding sequence of the enzyme EcoRV is the subsequence GATATC. How many exact matches has Chromosome I of Cel- egans. How many do you expect by chance? 9. Plot of codon usage. Go to the seqinr help page on dotchart.uco. (a) Redo the example and brieﬂy describe its usage. (b) Use the query language to ﬁnd 192 CHAPTER 9. ANALYZING SEQUENCES Chapter 10 Markov Models The idea of a Markov process forms the basis of many important models in bioinformatics such as (Hidden) Markov Models, models for sequence align- ment, and models for phylogenetic trees. By the latter it is possible to estimate distances between several sequences and to visualize these in a tree. Classical matrices for sequence alignment such as BLOSUM and PAM are constructed on the basis of a Markov process. By (Hidden) Markov Mod- els the speciﬁc repetitive order of DNA sequences can be modeled so that predictions of families becomes possible. In this chapter you learn what a probability transition matrix is and which role it plays in a Markov process to construct speciﬁc sequences. Various models for phylogenetic trees are explained in terms of the rate matrix as well as the probability transition matrix. The basic ideas of the Hidden Markov Model are brieﬂy explained and illustrated by an example 1 . 10.1 Random sampling Models to predict and classify DNA type of sequences make it possible to draw a sample from a population. The latter is the same as a distribution with certain properties. Recall from Chapter 3 that a discrete distribution is a set of values with certain probabilities that add up to one. Two basic examples illustrate this point. 1 This chapter is somewhat more technical in its notation with respect to e.g. conditional probability. This is, however, inevitable for the understanding of Markov processes. 193 194 CHAPTER 10. MARKOV MODELS Example 1. Throwing a coin. A fair coin X attains Head and Tail with probability 1/2. Thus we may write P(X = H) = 0.5 and P(X = T) = 0.5. With such a random variable there always correspond a population as well as a sampling scheme which can be simulated on a computer (e.g. Press, et al., 1992). > sample(c("H","T"),30,rep=TRUE,prob=c(0.5,0.5)) [1] "H" "H" "T" "T" "T" "H" "H" "T" "T" "H" "H" "H" "T" "T" "H" "T" [20] "H" "T" "T" "T" "H" "T" "H" "T" "T" "T" "T" Thus the sampled values Head and Tail correspond to the process of actu- ally throwing with a fair coin. The function sample randomly draws thirty times one of the values c("H","T") with replacement (rep=TRUE) and equal probabilities (prob=c(0.5,0.5)). Example 2. Generating a sequence of nucleotides. Another example is that of a random variable X which has the letters of the nucleotides as its values. So the events are X = A, X = C, X = G, and X = T. These events may occur in a certain DNA sequence with probabilities P(X = A) = 0.1, P(X = G) = 0.4, P(X = C) = 0.4, and P(X = T) = 0.1, respectively. Then the actual placement of the nucleotides along a sequence can be simulated. > sample(c("A","G","C","T"),30,rep=TRUE,prob=c(0.1,0.4,0.4,0.1)) [1] "G" "C" "T" "G" "C" "G" "G" "G" "T" "C" "T" "T" "C" "C" "C" [20] "G" "G" "C" "G" "G" "G" "C" "C" "C" "G" "C" Of course, if you do this again, then the resulting sequence will diﬀer due to the random nature of its generation. For these sampling schemes it holds that the events occur independently from the previous. 10.2 Probability transition matrix In order to build a model that produces speciﬁc sequences we will consider a certain type of random variable. In particular, we will consider a sequence {X 1 , X 2 , · · · } with values from a certain state space E. The latter is simply a set containing the possible values or states of a process. If, for instance, X n = i, then the process is in state i at time n. Similarly, the expression 10.2. PROBABILITY TRANSITION MATRIX 195 P(X 1 = i) denotes the probability that the process is in state i at time point 1. The event that the process changes its state from i to j (transition) between time point one and two corresponds to the event (X 2 = j|X 1 = i), where the bar means ”given that”. The probability for this event to happen is denoted by P(X 2 = j|X 1 = i). In general, the probability of the transition from i to j between time point n and n +1 is given by P(X n+1 = j|X n = i). These probabilities can be collected in a probability transition matrix P with elements p ij = P(X n+1 = j|X n = i). We will assume that the transition probabilities are the same for all time points so that there is no time index needed on the left hand side. Given that the process X n is in a certain state, the corresponding row of the tran- sition matrix contains the distribution of X n+1 , implying that the sum of the probabilities over all possible states equals one. The probability transition matrix contains a (conditional) discrete probability distribution on each of its rows. For a Markov process it holds that the state at time point n+1 de- pends upon the state at time point n, but not on states at earlier time points. Example 1. Using the probability transition matrix to generate a Markov sequence. Suppose X n has two states: 1 for a pyrimidine and 2 for a purine. A sequence can now be generated, as follows. If X n = 1, then we throw with a fair die: If the outcome is lower than or equal to 5, then X n+1 = 1 and, otherwise, (outcome equals 6) X n+1 = 2. If X n = 2, then we throw with a fair coin: If the outcome equals Tail, then X n+1 = 1, and otherwise X n+1 = 2. For this process the two by two probability transition matrix equals from to 1 2 1 p 11 p 12 2 p 21 p 22 , where p 21 is the probability that the process changes from 2 to 1. This transition matrix can also be written as P = _ p 11 p 12 p 21 p 22 _ = _ P(X 1 = 1|X 0 = 1) P(X 1 = 2|X 0 = 1) P(X 1 = 1|X 0 = 2) P(X 1 = 2|X 0 = 2) _ = _ 5 6 1 6 1 2 1 2 _ . Any matrix probability transition matrix P can be visualized by a transi- tion graph, where the transition probabilities are visualized by an arrow from 196 CHAPTER 10. MARKOV MODELS state i to state j and the value of p ij . For the current example the transition graph is given by Figure 10.1 2 . The values 1 and 2 of the process are written within the circles and the transition probabilities are written near the arrows. To actually generate a sequences with values equal to 1 and 2 according the 5/6 1/6 1/2 1/2 0 1 Figure 10.1: Graph of probability transition matrix transition matrix we may use the following. markov1 <- function(x,P,n){ seq <- x for(k in 1:(n-1)){ seq[k+1] <- sample(x, 1, replace=TRUE, P[seq[k],])} return(seq) } P <- matrix(c(1/6,5/6,0.5,0.5), 2, 2, byrow=TRUE) rownames(P) <- colnames(P) <- StateSpace <- x <- c(1,2) > markov1(x,P,30) [1] 1 2 1 2 1 2 2 1 2 1 2 2 1 2 1 2 2 1 2 1 2 1 2 1 2 2 2 2 2 2 In the function markov1 the actual sampling is conducted by sample. We sample one element from the set containing 1 and 2 according to the prob- abilities in row seq[k] of the matrix P. This makes the probabilities of the states dependent on the corresponding row of the transition matrix. We con- veniently use the fact that R adds an element to the sequence; we do not have to declare its length on beforehand (although we could!). The sequence has a ﬁxed start at State 1 and thereafter the ﬁrst row in the probability transition matrix. Note that without the return command the function does 2 The values 1 and 2 are erroneously depicted as 0 and 1, respectively 10.2. PROBABILITY TRANSITION MATRIX 197 not give any output. Example 2. A sequence with a large frequency of C and G. To illustrate that certain probability transition matrices imply a large frequency of C and G residues, we use the following. markov2 <- function(StateSpace,P,pi0,n){ seq <- character(n) seq[1] <- sample(StateSpace, 1, replace=TRUE, pi0) for(k in 1:(n-1)){ seq[k+1] <- sample(StateSpace, 1, replace=TRUE, P[seq[k],])} return(seq) } P <- matrix(c( 1/6,5/6,0,0, 1/8,2/4,1/4,1/8, 0,2/6,3/6,1/6, 0,1/6,3/6,2/6),4,4,byrow=TRUE) rownames(P) <- colnames(P) <- StateSpace <- c("a","c","g","t") pi0 <- c(1/4,1/4,1/4,1/4) x <- markov2(StateSpace,P,pi0,1000) > table(x) x a c g t 72 409 378 141 The function starts with sampling just once from the distribution with equal probabilities pi0. It conveniently uses the the column and row names of the probability transition matrix for the sampling. The probabilities to go from ”a” or ”t” to ”c” or ”g” are large and as well as that to stay within ”c” or ”g”. From the frequency table it can be observed that the majority of residues are ”c” or ”g”. Example 3. A sequence with high phenylalanine frequency. Now it is possible to construct a sequence which produces the amino acid phenylalanine (F) with high probability. Recall that it is coded by the triple TTT or TTC. We use the function getTrans of the seqinr package to translate nucleotide triplets into amino acids. 198 CHAPTER 10. MARKOV MODELS pi0 <- c(1/4,1/4,1/4,1/4) P <- matrix(c(.01,.01,.01,.97, .01,.01,.01,.97, .01,.01,.01,.97, .01,.28,.01,0.70),4,4,byrow=T) rownames(P) <- colnames(P) <- StateSpace <- c("a","c","g","t") x <- markov2(StateSpace,P,pi0,30000) > table(getTrans(x)) * A C D F H I L M N P R S T V W 2 1 75 2 5205 24 76 2260 1 2 19 26 2154 1 91 1 Y 60 From the table it is clear that the F frequency is the largest among the gen- erated amino acids. Example 4. To illustrate estimation of the probability transition matrix we proceed with the sequence produced by the previous example. nr <- count(x,2) names(nr) A <- matrix(NA,4,4) A[1,1]<-nr["aa"]; A[1,2]<-nr["ag"]; A[1,3]<-nr["ac"]; A[1,4]<-nr["at"] A[2,1]<-nr["ga"]; A[2,2]<-nr["gg"]; A[2,3]<-nr["gc"]; A[2,4]<-nr["gt"] A[3,1]<-nr["ca"]; A[3,2]<-nr["cg"]; A[3,3]<-nr["cc"]; A[3,4]<-nr["ct"] A[4,1]<-nr["ta"]; A[4,2]<-nr["tg"]; A[4,3]<-nr["tc"]; A[4,4]<-nr["tt"] rowsumA <- apply(A, 1, sum) Phat <- sweep(A, 1, rowsumA, FUN="/") rownames(Phat) <- colnames(Phat) <- c("a","g","c","t") > round(Phat,3) a g c t a 0.011 0.000 0.007 0.982 g 0.017 0.003 0.010 0.969 c 0.010 0.011 0.012 0.967 t 0.009 0.009 0.279 0.703 The number of transitions are counted and divided by the row totals. The estimated transition probabilities are quite close to the true transition proba- bilities. The zero transition probabilities are exactly equal to the true because 10.3. PROPERTIES OF THE TRANSITION MATRIX 199 these do not occur. This estimation procedure can easily be applied to DNA sequences. 10.3 Properties of the transition matrix In the above, the sequence was started at a certain state. Often, however, the probabilities of the initial states are available. That is, we have a vec- tor π 0 with initial probabilities π 10 = P(X 0 = 1) and π 20 = P(X 0 = 2). Furthermore, if the transition matrix P = _ p 11 p 12 p 21 p 22 _ = _ P(X 1 = 1|X 0 = 1) P(X 1 = 2|X 0 = 1) P(X 1 = 1|X 0 = 2) P(X 1 = 2|X 0 = 2) _ , then the probability that the process is in State 1 at time point 1 can be written as P(X 1 = 1) = π 10 p 11 + π 20 p 21 = π T 0 p 1 , (10.1) where p 1 is the ﬁrst column of P, see Section 10.7. Note that the last equality holds by deﬁnition of matrix multiplication. In a similar manner, it can be shown that P(X 1 = 2) = π T 0 p 2 , where p 2 is column 2 of the transition matrix P = (p 1 , p 2 ). It can be concluded that π T 0 P = π T 1 , where π T 1 = (P(X 1 = 1), P(X 1 = 2)); the probability at time point 1 that the process is in State 1, State 2, respectively. This holds in general for all time points n, that is π T n P = π T n+1 . (10.2) Thus to obtain the probabilities of the states at time point n + 1, we can simply use matrix multiplication 3 . Example 1. Matrix multiplication to compute probabilities. Suppose the following initial distribution and probability matrix π 0 = _ 2 3 1 3 _ , P = _ 5 6 1 6 1 2 1 2 _ , for State 1 and 2, respectively. Then P(X 1 = 1) and P(X 1 = 2) collected in π T 1 = (P(X 1 = 1), P(X 1 = 2)) can be computed as follows. π T 1 = π T 0 P = _ 2 3 1 3 _ _ 5 6 1 6 1 2 1 2 _ = _ 2 3 · 5 6 + 1 3 · 1 2 2 3 · 1 6 + 1 3 · 1 2 _ = _ 13 18 5 18 _ 3 The transposition sign T simply transforms a column into a row. 200 CHAPTER 10. MARKOV MODELS Using R its operator %*% for matrix multiplication, the product π T 0 P can be computed as follows. > P <- matrix(c(5/6,1/6,0.5,0.5),2,2,byrow=T) > pi0 <- c(2/3,1/3) > pi0 %*% P [,1] [,2] [1,] 0.7222222 0.2777778 Yet, another important property of the probability transition matrix deals with the probability of being in state 1 given that the process is in state 1 two time points before. In particular, it holds (see Section 10.7) that P(X 2 = 1|X 0 = 1) = p 2 11 , (10.3) where the latter is element (1, 1) of the matrix 4 P 2 . In general, we have that P(X n = j|X 0 = i) = p n ij , which is element i, j of P n . Example 3. Given the probability matrix of the previous example, the values P(X 2 = j|X 0 = i) for all of i, j can be computed by matrix multipli- cation. P 2 = _ 5 6 1 6 1 2 1 2 _ · _ 5 6 1 6 1 2 1 2 _ = _ ( 5 6 ) 2 + 1 6 1 2 5 6 1 6 + 1 6 1 2 1 2 5 6 + ( 1 2 ) 2 1 2 1 6 + ( 1 2 ) 2 _ = _ 28 36 8 36 24 36 12 36 _ . Obviously, such matrix multiplications can be accomplished much more con- venient on a personal computer. > P %*% P [,1] [,2] [1,] 0.7777778 0.2222222 [2,] 0.6666667 0.3333333 Larger powers of P can be computed more eﬃciently by methods given be- low. 4 For a brief deﬁnition of matrix multiplication, see Pevsner (2003, p.56) or wikipedia using the search string ”wiki matrix multiplication”. 10.4. STATIONARY DISTRIBUTION 201 10.4 Stationary distribution A probability distribution π satisfying π T = π T P is stationary because the transition matrix does not change the probabilities of the states of the process. Such a distribution usually exists, is unique, and plays an essential role in the long term behavior of the process. It sheds light on the question: What is the probability P(X n = 1|X 0 = 1) = p n 11 , as n increases without bound. That is: What is the probability that the process is in State 1, given that it started in State 1, as time increases without bound? To answer such a question we need large powers of the probability transition matrix. To compute these we need the eigen-decomposition of the probability transition matrix P = V ΛV −1 , where V is the eigenvector matrix and Λ the diagonal matrix with eigen- values. The latter are usually sorted in decreasing order so that the ﬁrst (left upper) is the largest. Now the third power of the probability transition matrix can be computed, as follows P 3 = V ΛV −1 V ΛV −1 V ΛV −1 = V ΛΛΛV −1 = V Λ 3 V −1 . So that, indeed, in general P n = V Λ n V −1 . The latter is a computationally convenient expression because we only have to take the power of the eigenvalues in Λ and to multiply by the left and right eigenvector matrices. This will be illustrated below. In the long term the Markov process tends to a certain value (Br´emaud, 1999, p.197) because a probability transition matrix has a unique largest eigenvalue equal to 1 with corresponding eigenvectors 1 and π (or rather normalized versions of these). It follows that, as n increases without bound, then P n tends to 1π T . In other words, P(X n = j|X 0 = i) = p n ij tends to element (i, j) of 1π T , which is equal to element j of π. For any initial dis- tribution π 0 , it follows that π 0 P n tends to π T . Example 1. Stationary distribution. To compute the eigen-decomposition of the probability transition matrix P as well as powers of it, we may use the function eigen. 202 CHAPTER 10. MARKOV MODELS > P <- matrix(c(1/6,5/6,0.5,0.5),2,2,byrow=T) > V <- eigen(P,symmetric = FALSE) > Vvalues
[1] 1.0000000 -0.3333333
> V$vectors [,1] [,2] [1,] -0.7071068 -0.8574929 [2,] -0.7071068 0.5144958 The output of the function eigen is assigned to the list V from which the eigenvalues and eigenvectors can be extracted and printed to the screen. Now we can compute P 16 ; the probability transition matrix raised to the power sixteen. > V$vec %*% diag(V$va)^(16) %*% solve(V$vec)
[,1] [,2]
[1,] 0.375 0.625
[2,] 0.375 0.625
So that the stationary distribution π
T
equals (0.375, 0.625).
Example 2. Diploid. Suppose A is a dominant gene, a a recessive
and that we start with a heterozygote aA. From the latter we obtain the
initial state probability π
T
= (0, 1, 0) for the events (AA, aA, aa). When
we consider pure self-fertilization, then the oﬀspring from AA is AA with
probability (1, 0, 0), that of aa is aa with probability (0, 0, 1), and that of
aA is (AA, aA, aa) with probability 1/4, 1/2, 1/4, respectively. Hence, the
probability transition matrix becomes
P =
_
_
1 0 0
1/4 1/2 1/4
0 0 1
_
_
We can now compute the transition probability matrix after ﬁve generations.
P <- matrix(c(1,0,0, 1/4,1/2,1/4,0,0,1),3,3,byrow=T)
V <- eigen(P,symmetric = FALSE)
> V$vec %*% diag(V$va)^(5) %*% solve(V$vec) [,1] [,2] [,3] [1,] 1.000000 0.00000 0.000000 10.5. PHYLOGENETIC DISTANCE 203 [2,] 0.484375 0.03125 0.484375 [3,] 0.000000 0.00000 1.000000 Hence, the distribution we obtain can be read from the second row which is highly homozygotic. A little more precise, using Equation 10.2, it can be shown that π T n+1 = _ 1 2 _ 1 2 _ n , _ 1 2 _ n , 1 2 _ 1 2 _ n _ , so that the distribution converges to (1/2, 0, 1/2). Note that this method of raising the transition probability matrix to a large power can easily be applied to determine the stationary distribution. The idea of taking a transition matrix to a certain power is also used to construct the PAM250 matrix given the PAM1 matrix (Pevsner, 2003, p.53) and for the construction of various BLOSUM matrices (Pevsner, 2003, p.50- 59; Deonier, et al. 2005, 187-190). 10.5 Phylogenetic distance Phylogenetic trees are constructed on the basis of distances between DNA sequences. These distances are computed from substitution models which are deﬁned by a matrix representing the rate of substitutions of one state to the other. The latter is usually expressed as a matrix Q. The rates of staying in a state are given by a negative number on the diagonal of the substitution matrix. The probability transition matrix P can be computed by matrix exponentiation P = exp(Q). How to do this in practice will be illustrated by an example. Example 1. From a rate matrix to a probability transition matrix. Suppose the rate matrix Q = A G C T A G C T _ ¸ ¸ _ −0.60 0.20 0.20 0.20 0.20 −0.60 0.20 0.20 0.20 0.20 −0.60 0.20 0.20 0.20 0.20 −0.60 _ ¸ ¸ _ . Thus within a certain time period a proportion of 0.20 A changes into G, 0.20 A into C, and 0.20 A into T. Consequently, a proportion of 0.60 of the 204 CHAPTER 10. MARKOV MODELS residues goes back to A. Given this rate matrix, we can ﬁnd the probabil- ity transition matrix P = exp(Q) by using the function expm(Q) from the package Matrix. library(Matrix) Q <- 0.2 * Matrix(c(-3,1,1,1,1,-3,1,1,1,1,-3,1,1,1,1,-3),4) rownames(Q) <- colnames(Q) <- c("A","G","C","T") P <- as.matrix(expm(Q)) > round(P,2) A G C T A 0.59 0.14 0.14 0.14 G 0.14 0.59 0.14 0.14 C 0.14 0.14 0.59 0.14 T 0.14 0.14 0.14 0.59 Thus the probability that the state changes from A to A is 0.59, from A to G is 0.14, etc. Because all phylogenetic models are deﬁned in terms of rate matrices, we shall concentrate on these. For instance, the rate matrix for the Jukes and Cantor (1969) (JC69) model can be written as Q JC69 = A G C T A G C T _ ¸ ¸ _ · α α α α · α α α α · α α α α · _ ¸ ¸ _ . The sum of each row of a rate matrix equals zero, so that from this require- ment the diagonal elements of the JC69 model are equal to −3α. Further- more, the non-diagonal substitution rates of the JC69 model all have the same value α. That is, the change from i to j equals that from j to i, so that the rate matrix is symmetric. Also the probability that the sequence equals one of the nucleotides is 1/4. This assumption, however, is unrealistic is many cases. Transitions are substitutions of nucleotides within types of nucleotides, thus purine to purine or pyrmidine to pyrmidine (A ↔ G or C ↔ T). Transversions are substitutions between nucleotide type (A ↔ T, G ↔ T,A ↔ C, and C ↔ G). In the JC69 model a transition is assumed to 10.5. PHYLOGENETIC DISTANCE 205 happen with equal probability as a transversion. That is, it does not account for the fact that transitions are more common that transversions. To cover this for more general type of models are proposed by Kimura (1980, 1981), which are commonly abbreviated by K80 and K81. In terms of the rate matrix these models can we written as Q K80 = _ ¸ ¸ _ · α β β α · β β β β · α β β α · _ ¸ ¸ _ , Q K81 = _ ¸ ¸ _ · α β γ α · γ β β γ · α γ β α · _ ¸ ¸ _ . In the K80 model a change within type (transition) occurs at rate α and between type (transversion) at rate β. In the K81 model all changes occur at a diﬀerent though symmetric rate; the rate of change A → G is α and equals that of A ←G. If α is large, then the amount of transitions is large; if both β and γ are very small, then the number of transversions is small. A model is called “nested” if it is a special case of a more general model. For instance, the K80 model is nested in the K81 model because when we take γ = β, then we obtain the K80 model. Similarly, the JC69 model is nested in the K80 model because if we take β = α, then we obtain the JC69 model. Some examples of models with even more parameters are the Hasegawa, Kishino, and Yano (1985) (HKY85) model and the General Time-Reversable Model (GTR) model Q HKY 85 = _ ¸ ¸ _ · απ G βπ C βπ T απ A · βπ C βπ T βπ A βπ G · απ T βπ A βπ G απ C · _ ¸ ¸ _ , Q GTR = _ ¸ ¸ _ · απ G βπ C γπ T απ A · δπ C π T βπ A δπ G · ζπ T γπ A π G ζπ C · _ ¸ ¸ _ . The distance between DNA sequences is deﬁned on the basis of these models. From these distances the phylogenetic tree is computed by a neighbor-joining algorithm such that it has the smallest total branch length. Example 2. The K81 model. To compute the rate matrix of the K81 model with α = 3/6, β = 2/6, γ = 1/6 we may use the following. alpha <- 3/6; beta <- 2/6; gamma<- 1/6; Q <- matrix(data=NA,4,4) Q[1,2] <- Q[2,1] <- Q[3,4] <- Q[4,3] <- alpha 206 CHAPTER 10. MARKOV MODELS Q[1,3] <- Q[3,1] <- Q[2,4] <- Q[4,2] <- beta Q[1,4] <- Q[4,1] <- Q[2,3] <- Q[3,2] <- gamma > diag(Q) <- -(alpha + beta + gamma) > Q [,1] [,2] [,3] [,4] [1,] -1.0000000 0.5000000 0.3333333 0.1666667 [2,] 0.5000000 -1.0000000 0.1666667 0.3333333 [3,] 0.3333333 0.1666667 -1.0000000 0.5000000 [4,] 0.1666667 0.3333333 0.5000000 -1.0000000 > Q <- Matrix(Q) > P <- as.matrix(expm(Q)) > P [,1] [,2] [,3] [,4] [1,] 0.4550880 0.2288517 0.1767105 0.1393498 [2,] 0.2288517 0.4550880 0.1393498 0.1767105 [3,] 0.1767105 0.1393498 0.4550880 0.2288517 [4,] 0.1393498 0.1767105 0.2288517 0.4550880 By raising the power of the probability transition matrix to a suﬃciently large number, it can be observed that the stationary distribution π T = (0.25, 0.25, 0.25, 0.25). Example 3. Stationarity for the JC69 model. Let’s take α = 1/5 as in Example 1 and compute the rate matrix Q of the JC69 model, the corresponding probability transitionmatrix P, and raise it to the power 50. library(Matrix) alpha <- 1/5; Q <- matrix(rep(alpha,16),4,4) diag(Q) <- -3 * alpha Q <- Matrix(Q) P <- as.matrix(expm(Q)) V <- eigen(P,symmetric = FALSE) > V$vec %*% diag(V$va)^(50) %*% solve(V$vec)
[,1] [,2] [,3] [,4]
[1,] 0.25 0.25 0.25 0.25
[2,] 0.25 0.25 0.25 0.25
[3,] 0.25 0.25 0.25 0.25
[4,] 0.25 0.25 0.25 0.25
10.5. PHYLOGENETIC DISTANCE 207
Hence, the stationary distribution is π
T
= (0.25, 0.25, 0.25, 0.25) (cf. Ewens
& Grant, 2005, p. 477).
Example 4. Distance between two sequences according to the JC69
model. In case of the JC69 model, the distance between sequences is a
function of the proportion of diﬀerent nucleotides. Namely,
d = −
3
4
log(1 −4p/3),
where p is the proportion of diﬀerent nucleotides of the two sequences. The
pairwise distances between DNA sequences can be computed by the function
dist.dna from the ape package.
> library(ape);library(seqinr)
> accnr <- paste("AJ5345",26:27,sep="")
> seqbin <- read.GenBank(accnr, species.names = TRUE, as.character = FALSE)
> dist.dna(seqbin, model = "JC69")
AJ534526
AJ534527 0.1326839
Hence, the distance is 0.133. Over a total of 1143 nucleotides there are 139
diﬀerences, som that the proportion of diﬀerent nucleotides 139/1143 = p.
Inserting this into the previous distance formula gives the distance. This can
be veriﬁed as follows.
> seq <- read.GenBank(accnr, species.names = TRUE, as.character = TRUE)
> p <- sum(seq$AJ534526==seq$AJ534527)/1143
> d <- -log(1-4*p/3)*3/4
> d
[1] 0.1326839
Chamaea fasciata mitochondrial cytb gene for cytochrome b for 10 species of
warblers of the genus sylvia (Paradis, 2006). The function paste is used to
quickly deﬁne the accession numbers and read.GenBank to actually down-
load the sequences. The species names are extracted and attached to the
sequences. We shall use the dist.dna function with the K80 model.
208 CHAPTER 10. MARKOV MODELS
library(ape);library(seqinr)
accnr <- paste("AJ5345",26:35,sep="")
names(seq) <- attr(seq, "species")
dist <- dist.dna(seq, model = "K80")
plot(nj(dist))
Obviously, in this manner various trees can be computed and their plots
compared.
When various diﬀerent models are deﬁned the question becomes appar-
ent which of these ﬁts best to the data relative to the number of parameters
(symbols) of the model. When the models are estimated by maximum likeli-
hood, then the Akaike information criterion (AIC = -2 · loglik + 2 · number
of free parameters) can be used to select models. The best model is the one
with the smallest AIC value.
Example 6. A program called PHYML (Guindon & Gascuel, 2003)
function phymltest, if the executable is available at the same directory. We
ﬁrst write the sequences to the appropriate directory. The output from the
program is written to the object called out for which the functions plot(out)
and summary(out) can be used to extract more detailed information.
> setwd("/share/home/wim/bin")
> write.dna(seq,"seq.txt", format ="interleaved")
> out <-phymltest("seq.txt",format = "interleaved", execname ="phyml_linux")
> print(out)
nb.free.para loglik AIC
JC69 1 -4605.966 9213.931
JC69+I 2 -4425.602 8855.203
JC69+G 2 -4421.304 8846.608
JC69+I+G 3 -4421.000 8848.001
K80 2 -4423.727 8851.455
K80+I 3 -4230.539 8467.079
K80+G 3 -4224.457 8454.915
K80+I+G 4 -4223.136 8454.272
F81 4 -4514.331 9036.662
10.6. HIDDEN MARKOV MODELS 209
F81+I 5 -4309.600 8629.199
F81+G 5 -4304.530 8619.060
F81+I+G 6 -4303.760 8619.519
F84 5 -4351.164 8712.328
F84+I 6 -4112.006 8236.012
F84+G 6 -4106.568 8225.135
F84+I+G 7 -4105.500 8225.001
HKY85 5 -4333.086 8676.171
HKY85+I 6 -4102.262 8216.524
HKY85+G 6 -4097.401 8206.802
HKY85+I+G 7 -4096.624 8207.248
TN93 6 -4323.291 8658.581
TN93+I 7 -4097.099 8208.198
TN93+G 7 -4091.461 8196.922
TN93+I+G 8 -4090.790 8197.580
GTR 9 -4293.398 8604.795
GTR+I 10 -4084.522 8189.043
GTR+G 10 -4079.010 8178.020
GTR+I+G 11 -4078.149 8178.299
The notation ”+I” and ”+G” indicates the presence of invariant sites and/or
a gamma distribution of substitution rates. It can be seen that the smallest
AIC corresponds to model 27 called GTR+G. To plot it, we have to read the
trees, and, next, to extract the 27th, see Figure 10.3.
plot(tr[[27]])
In case similar sequences have slightly diﬀerent lengths, these have to be
aligned by programs such as clustalx or clustalw before these can be
used.
10.6 Hidden Markov Models
In a Hidden Markov Model (HMM) there are two probability transition ma-
trices. There is an emission matrix E and a transition matrix A. The
210 CHAPTER 10. MARKOV MODELS
generation of an observable sequence goes in two steps. First, there is a tran-
sition from a Markov process of a hidden state and given this value there
is an emission of an observable value. We shall illustrate this by the clas-
sical example of the occasionally dishonest casino (Durbin et. al., 1998, p.18).
Example 1. Occasionally dishonest casino. A casino uses a fair die most
of the time, however, occasionally it switches to an unfair die. The state with
respect to fairness is hidden for the observer. The observer can only observe
the values of the die and not its hidden state with respect to its fairness. It
is convenient to denote fair by 1 and unfair by 2. The transition probabilities
of the hidden states are by the emission matrix
E =
_
P(D
i
= 1|D
i−1
= 1) P(D
i
= 2|D
i−1
= 1)
P(D
i
= 1|D
i−1
= 2) P(D
i
= 2|D
i−1
= 2)
_
=
_
0.95 0.05
0.10 0.90
_
.
Thus the probability is 0.95 that the die is fair at time point i, given that
it is fair at time point i −1. The probability that it will switch from fair to
unfair is 0.05. The probability that it will switch from loaded to fair is 0.10
and that it stays loaded is 0.90. With this emission matrix we can generate a
sequence of hidden states, where the values 1 and 2 indicate whether the die
is fair (1) or loaded (2). Given the fairness of the die we deﬁne the probability
transition matrix.
A =
_
P(O
i
= 1|D
i
= 1) P(O
i
= 2|D
i
= 1) P(O
i
= 3|D
i
= 1) · · ·
P(O
i
= 1|D
i
= 2) P(O
i
= 2|D
i
= 2) P(O
i
= 3|D
i
= 2) · · ·
_
=
_
1/6 1/6 1/6 1/6 1/6 1/6
1/10 1/10 1/10 1/10 1/10 1/2
_
. (10.4)
Thus given that the die is fair, the probability of any outcome equals 1/6.
However, given that the die is unfair (loaded), the probability of outcome 6
equals 1/2 and that of any other outcome equals 1/10.
The HMM with this transition and emission matrix can be programmed.
After sampling the hidden states from a Markov chain and the outcomes of
the die are sampled according to the value of the hidden state (die type).
hmmdat <- function(A,E,n){
observationset <- c(1:6)
hiddenset <- c(1,2)
x <- h <- matrix(NA,nr=n,nc=1)
10.6. HIDDEN MARKOV MODELS 211
h[1]<-1; x[1]<-sample(observationset,1,replace=T,E[h[1],])
h <- markov(hiddenset,A,n)
for(k in 1:(n-1)){x[k+1] <- sample(observationset,1,replace=T,E[h[k],])}
out <- matrix(c(x,h),nrow=n,ncol=2,byrow=FALSE)
return(out)
}
E <- matrix(c(rep(1/6,6),rep(1/10,5),1/2),2,6,byrow=T) #emission matrix
A <- matrix(c(0.95,0.05,0.1,0.9),2,2,byrow=TRUE) #transition matrix
dat <- hmmdat(A,E,100)
colnames(dat) <- c("observation","hidden_state")
rownames(dat) <- 1:100
> t(dat)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
observations 5 2 3 1 6 1 3 1 1 5 6 6 2 2 3 5 4 6 1 2 4 4 3 2 3
hidden_states 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47
observations 4 3 2 4 1 6 6 6 6 6 5 5 3 6 1 6 5 2 4 1 4 2
hidden_states 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1
48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69
observations 5 6 5 2 3 3 1 3 3 5 6 6 2 4 5 4 6 1 6 5 2 6
hidden_states 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
observations 1 1 4 4 1 5 6 4 3 5 4 2 6 1 3 6 5 2 2 6 6 1
hidden_states 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 1 1
92 93 94 95 96 97 98 99 100
observations 4 1 6 5 5 6 5 3 4
hidden_states 1 1 1 1 1 1 1 1 1
In certain applications to bioinformatics, it is of most importance to es-
timate the value of the hidden state given the data. The Viterbi algorithm
is developed to predict the hidden state given the data and the (estimated)
transition and emission matrix. The algorithm builds up a matrix v(i, l),
where i runs from one to the number of observations and l from one to the
number of states. The initial values are v(1, 1) = 1, and v(1, l) = 0 for all l.
Then the values for v(i, l) are recursively deﬁned by
v(i, l) = e(l, x(i)) · max
k
{v(i −1, k)a(k, l)} .
212 CHAPTER 10. MARKOV MODELS
For each row of the matrix the maximum is taken as the best predictor of
the hidden state.
Example 2. The viterbi algorithm can be programmed and applied
to the hidden states of the data generated with respect to the occasionally
dishonest casino.
viterbi <- function(A,E,x) {
v <- matrix(NA, nr=length(x), nc=dim(A)[1])
v[1,] <- 0; v[1,1] <- 1
for(i in 2:length(x)) {
for (l in 1:dim(A)[1]) {v[i,l] <- E[l,x[i]] * max(v[(i-1),] * A[l,])}
}
return(v)
}
vit <- viterbi(A,E,dat[,1])
vitrowmax <- apply(vit, 1, function(x) which.max(x))
hiddenstate <- dat[,2]
> table(hiddenstate, vitrowmax)
vitrowmax
hiddenstate 1 2
1 72 11
2 15 2
datt <- cbind(dat,vitrowmax)
colnames(datt) <- c("observation","hidden_state","predicted state")
> t(datt)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
observation 5 2 3 1 6 1 3 1 1 5 6 6 2 2 3 5 4 6 1 2 4 4 3 2
hidden_state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
predicted state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
observation 3 4 3 2 4 1 6 6 6 6 6 5 5 3 6 1 6 5 2 4 1
hidden_state 1 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1
predicted state 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
observation 4 2 5 6 5 2 3 3 1 3 3 5 6 6 2 4 5 4 6 1 6
hidden_state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
10.7. APPENDIX 213
predicted state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87
observation 5 2 6 1 1 4 4 1 5 6 4 3 5 4 2 6 1 3 6 5 2
hidden_state 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2
predicted state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
88 89 90 91 92 93 94 95 96 97 98 99 100
observation 2 6 6 1 4 1 6 5 5 6 5 3 4
hidden_state 2 2 1 1 1 1 1 1 1 1 1 1 1
predicted state 1 1 1 1 1 1 1 1 1 1 1 1 1
The misclassiﬁcation rate is 0.27 which is quite large given the fact that we
used the true transition and emission matrix. An important observation is
that after a transition of a hidden state, it takes a few values for the predic-
tion to change. This is caused by the recursive nature of the algorithm.
10.7 Appendix
The probability that the process is in State 1 at time point 1 can be computed
as follows.
P(X
1
= 1) = P(X
1
= 1, X
0
= 1) + P(X
1
= 1, X
0
= 2)
= P(X
1
= 1|X
0
= 1) · P(X
0
= 1) + P(X
1
= 1|X
0
= 2) · P(X
0
= 2)
= π
10
p
11
+ π
20
p
21
= π
T
0
p
1
,
where p
1
is the ﬁrst column of P.
214 CHAPTER 10. MARKOV MODELS
In particular, it holds that
P(X
2
= 1|X
0
= 1) = P(X
2
= 1, X
1
= 1|X
0
= 1) + P(X
2
= 1, X
1
= 2|X
0
= 1)
=
2

k=1
P(X
2
= 1, X
1
= k|X
0
= 1)
=
2

k=1
P(X
2
= 1|X
1
= k, X
0
= 1) · P(X
1
= k|X
0
= 1)
=
2

k=1
P(X
2
= 1|X
1
= k) · P(X
1
= k|X
0
= 1)
= p
11
p
11
+ p
21
p
12
= row 1 of P times column 1 of P = P
2
11
,
where the latter is element (1, 1) of the matrix P
2
= P · P.
10.8 Overview and concluding remarks
The probability transition matrix is extensively explained and illustrated
because it is a cornerstone to many ideas in bioinformatics. A thorough
treatment of phylogenetics is given by Paradis (2006) and of Hidden Markov
Models by Durbin et. al (2005).
10.9 Exercises
1. Visualize by a transition graph the following transition matrices. For
the process with four states take the names of the nucleotides in the
order A, G, T, and C.
_
1
3
2
3
3
4
1
4
_
,
_
1 0
0 1
_
,
_
0 1
1 0
_
,
_
_
_
_
1
4
2
4
0
1
4
1
6
2
6
2
6
1
6
0
2
7
5
7
0
1
8
1
8
2
8
4
8
_
_
_
_
,
_
_
_
_
1
4
3
4
0 0
1
6
5
6
0 0
0 0
5
7
2
7
0 0
3
8
5
8
_
_
_
_
.
2. Computing probabilities. Given the states 0 and 1 and the following
initial distribution and probability matrix
π
0
=
_
1
2
1
2
_
, P =
_
3
4
1
4
1
2
1
2
_
.
10.9. EXERCISES 215
(a) Compute P(X
1
= 0).
(b) Compute P(X
1
= 1).
(c) Compute P(X
2
= 0|X
0
= 0).
(d) Compute P(X
2
= 1|X
0
= 0).
3. Programming GTR. Use π
A
= 0.15, π
G
= 0.35, π
C
= 0.35, π
T
= 0.15,
α = 4, β = 0.5, γ = 0.4, δ = 0.3, = 0.2, and ζ = 4.
(a) Program the rate matrix in such a manner that it is simple to
adapt for other values of the parameters.
(b) Is the transversion rate larger or smaller then the transition rate?
(c) Compute the corresponding the probability transition matrix.
(d) Try to argue whether you expect a large frequency of transversions
or translations.
(e) Generate a sequence of 99 nucleotide residues according to the
markov model.
4. Distance according to JC69.
(a) Down load the sequences AJ534526 and AJ534527. Hint: Use
as.character = TRUE in the read.GenBank function.
(b) Compute the proportion of diﬀerent nucleotides.
(c) Use this proportion to verify the distances between these sequences
according to the JC69 model.
216 CHAPTER 10. MARKOV MODELS
Akaike information criterion for phymlout
8200
8400
8600
8800
9000
9200
GTR+Γ
GTR+ I +Γ
GTR+ I
TN93 +Γ
TN93 + I +Γ
HKY85 +Γ
HKY85 + I +Γ
TN93 + I
HKY85 + I
F84 + I +Γ
F84 +Γ
F84 + I
K80 + I +Γ
K80 +Γ
K80 + I
GTR
F81 +Γ
F81 + I +Γ
F81 + I
TN93
HKY85
F84
JC69 +Γ
JC69 + I +Γ
K80
JC69 + I
F81
JC69
Figure 10.2: Evaluation of models by AIC .
10.9. EXERCISES 217
Chamaea fasciata
Sylvia nisoria
Sylvia layardi
Sylvia subcaeruleum
Sylvia boehmi
Sylvia buryi
Sylvia lugens
Sylvia leucomelaena
Sylvia hortensis
Sylvia crassirostris
0.01
Figure 10.3: Tree according to GTR model.
218 CHAPTER 10. MARKOV MODELS
Appendix A
Answers to exercises of Chapter 1: Brief Introduction to R
1. Some questions to orientate yourself.
(a) matrix, numeric, numeric, matrix, function, function, factor, stan-
dardGeneric, ExpressionSet.
(b) remove, summation, product, sequence, standard deviation, num-
ber of rows,
(c) Use R its help or use the internet search key ”r wiki grep” to
ﬁnd the following answers: searching regular expressions, return
a vector from a function on the rows or columns of a matrix,
on packages, make R reading input from a ﬁle or URL, set the
working directory to a certain map, print the last · commands
given from the command line, give the structure of an object.
2. gendat
(a) apply(gendat,2,sd).
(b) apply(gendat,1,sd).
(c) To order the data frame according to the gene standard deviations.
sdexprsval <- apply(gendat,1,sd)
o <- order(sdexprsval,decreasing=TRUE)
gendat[o,]
219
220 APPENDIX A. ANSWERS TO EXERCISES
(d) gene1
3. Computations on gene means of the Golub data.
(a) Computation of mean gene expression values.
data(golub, package = "multtest")
meangol <- apply(golub,1,mean)
(b) To order the data frame use o <- order(meangol,decreasing=TRUE)
and golub[o,]
(c) Give the names of the three genes with the largest mean expression
value.
> golub.gnames[o[1:3],3]
[1] "U43901_rna1_s_at" "M13934_cds2_at" "X01677_f_at"
(d) Give their biological names.
> golub.gnames[o[1:3],2]
[1] "37 kD laminin receptor precursor/p40 ribosome associated protein gene"
[2] "RPS14 gene (ribosomal protein S14) extracted from Human ribosomal protein S14 gene"
[3] "GAPD Glyceraldehyde-3-phosphate dehydrogenase"
4. Computations on gene standard deviations of the Golub data.
(a) The standard deviation per gene can be computed by sdgol <-
apply(golub,1,sd).
(b) The gene with standard deviation larger than 0.5 can be selected
by golubsd <- golub[sdgol>0.5,].
(c) sum(sdgol>0.5) gives that the number of genes having sd larger
than 0.5 is 1498.
5. Oncogenes in Golub data.
(a) length(agrep("^oncogene",golub.gnames[,2])) gives 42.
(b) By the script below the "Cellular oncogene c-fos is found.
data(golub, package="multtest")
rowindex <- agrep("^oncogene",golub.gnames[,2])
oncogol <- golub[rowindex,]
221
oncogolub.gnames <- golub.gnames[rowindex,]
gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
meangol <- apply(oncogol[,gol.fac=="ALL"],1,mean)
o <- order(meangol,decreasing=TRUE)
> oncogolub.gnames[o[1:3],2]
[1] "PIM1 Pim-1 oncogene" "JUNB Jun B proto-oncogene"
[3] "Proto-oncogene BCL3 gene"
(c) meangol <- apply(oncogol[,gol.fac=="AML"],1,mean)
o <- order(meangol,decreasing=TRUE)
> oncogolub.gnames[o[1:3],2]
[1] "PIM1 Pim-1 oncogene" "JUNB Jun B proto-oncogene"
[3] "Proto-oncogene BCL3 gene"
(d) Writing results to a csv ﬁle. Be aware of the correct column
separation.
x <- oncogolub.gnames[o[1:10],c(3,2)]
colnames(x) <- c("probe ID","gene name")
write.csv(x,file="goluboutcsv")
write.table(x,file="goluboutnorowname",row.names=FALSE)
6. Constructing a factor.
(a) gl(2,4).
(b) gl(5,3).
(c) gl(3,5).
7. Gene means for B1 patients.
library(ALL); data(ALL)
meanB1 <- apply(exprs(ALL[,ALL$BT=="B1"]),1, mean) o <- order(meanB1,decreasing=TRUE) > meanB1[o[1:3]] AFFX-hum_alu_at 31962_at 31957_r_at 13.41648 13.16671 13.15995 Answers to exercises of Chapter 2: Descriptive Statistics and Data Dis- play 222 APPENDIX A. ANSWERS TO EXERCISES 1. Illustration of mean and standard deviation. (a) Use x<- c(1,1.5,2,2.5,3) and mean(x) and sd(x) to obtain the mean is 2 and the standard deviation is 0.7905694. (b) Now the mean is 7.4 and dramatically increased the standard de- viation 12.64615. (c) The outlier increased the mean as well as the standard deviation. 2. Comparing two genes. Take i <- 66 or i <- 790. (a) Use boxplot(golub[i,]~gol.fac) to observe that 790 has three outliers and 66 has no. (b) Use qqnorm(golub[i,gol.fac=="ALL") and qqline(golub[i,gol.fac=="ALL"]) to observe that nearly all values of 66 are on the line, where as for 790 the three outliers are way of the normality line. Hypothesis: The expression values of 66 are normally distributed, but those of row 790 are not. (c) Use mean(golub[i,gol.fac=="ALL"]) and median(golub[i,gol.fac=="ALL"]). The mean (-1.174024) is larger than the median (-1.28137) due to outliers on the right hand side. For the gen in row 66 the mean is 1.182503 and the median 1.23023. The diﬀerences are smaller. 3. Eﬀect size. (a) The size 11 is large, because the mean is eleven times larger than the standard deviation. data(golub, package="multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) efs <- apply(golub[,gol.fac=="ALL"],1,function(x) mean(x)/sd(x)) o <- order(efs,decreasing=TRUE) > efs[o[1:5]] [1] 11.138128 10.638308 9.155108 8.954115 8.695353 > golub.gnames[o[1:5],2] [1] "YWHAZ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide" [2] "ZNF91 Zinc finger protein 91 (HPF7, HTF10)" [3] "HnRNP-E2 mRNA" [4] "54 kDa protein mRNA" [5] "Immunophilin homolog ARA9 mRNA" 223 (b) The robust variant can be deﬁned by dividing the median by the MAD. An alternative would be to divide the median by the IQR. This gives other best genes indicating that the some genes may have outliers that inﬂuence the outcome. refs <- apply(golub[,gol.fac=="ALL"],1,function(x) median(x)/mad(x)) o <- order(refs,decreasing=TRUE) > refs[o[1:5]] [1] 14.51217 13.57425 13.27698 13.14419 12.91608 > golub.gnames[o[1:5],2] [1] "COX6B gene (COXG) extracted from Human DNA from overlapping chromosome 19 cosmids R31396, F25451, and R31076 containing COX6B and UPKA, genomic sequence" [2] "AFFX-HSAC07/X00351_M_at (endogenous control)" [3] "ATP5A1 ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit, isoform 1, cardiac muscle" [4] "ATP SYNTHASE GAMMA CHAIN, MITOCHONDRIAL PRECURSOR" [5] "YWHAZ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide" 4. Plotting gene expressions "CCND3 Cyclin D3". The answers in the script below. data(golub, package = "multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) stripchart(golub[1042,] ~ gol.fac,method="jitter") stripchart(golub[1042,] ~ gol.fac,method="jitter",vertical = TRUE) stripchart(golub[1042,] ~ gol.fac,method="jitter",col=c("red", "blue"), vertical = TRUE) stripchart(golub[1042,] ~ gol.fac,method="jitter",col=c("red", "blue"), pch="*",vertical = TRUE) title("CCND3 Cyclin D3 expression value for ALL and AMl patients") 5. Box-and-Whiskers plot of "CCND3 Cyclin D3".. locator() x11() x <- data(golub, package = "multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) boxplot(x,xlim=c(0,4)) 224 APPENDIX A. ANSWERS TO EXERCISES arrows(2.0,1.93,1.24,1.93);text(2.5,1.93,"Median") arrows(2.0,1.1,1.24,1.1) ;text(2.5,1.1,"Outlier") arrows(2.0,1.79,1.24,1.79);text(2.5,1.79,"first quartile") arrows(2.0,2.17,1.24,2.17);text(2.5,2.17,"third quartile") arrows(2.0,1.27,1.24,1.27);text(2.5,1.27,"lower wisker") arrows(2.0,2.59,1.24,2.59);text(2.5,2.59,"upper wisker") dev.copy2eps(device=x11,file="BoxplotWithExplanation.eps") boxplot.stats(x, coef = 1.5, do.conf = TRUE, do.out = TRUE) #finds values 6. Box-and-wiskers plot of persons of Golub et al. (1999) data.. (a) The medians are all around zero, the inter quartile range diﬀer only slightly, the minimal values are all around minus 1.5. All persons have outliers near three. (b) The means are very close to zero. The medians are all between (−0.15383, 0.06922), so these are also close to zero. personmean <- apply(golub,2,mean) personmedian <- apply(golub,2,median) (c) The data seem preprocessed to have standard deviation equal to one. The re-scaled IQR and MAD have slightly larger range. > range(apply(golub,2,sd)) [1] 0.9999988 1.0000011 > range(apply(golub,2,function(x) IQR(x)/1.349)) [1] 0.9599036 1.3361527 > range(apply(golub,2,mad)) [1] 0.9590346 1.2420185 7. Oncogenes of Golub et al. (1999) data. (a) Note that we need the transpose operator t to change rows into columns. The script below will do. data(golub, package="multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) rowindex <- agrep("^oncogene",golub.gnames[,2]) oncogol <- golub[rowindex,] oncogolub.gnames <- golub.gnames[rowindex,] 225 row.names(oncogol) <- oncogolub.gnames[,3] boxplot(data.frame(t(oncogol[,gol.fac=="ALL"]))) (b) The plot gives a nice overview of the distributions of the gene expressions values of the onco gene separately for the ALL and the AML patients. Several genes behave similarly for ALL and AML. Some are clearly distributed around zero, but others not. Also, some have a small inter quartile ranges, while for others this is large. A similar statement holds for outliers, some do not have outliers, but others certainly have. Some gene show distinct distributions between patient groups. For instance, the sixth has ALL expressions around zero, but those for AML are larger than zero. par(mfrow=c(2,1)) boxplot(data.frame(t(oncogol[,gol.fac=="ALL"]))) title("Box and wiskers plot for oncogenes of ALL patients ") boxplot(data.frame(t(oncogol[,gol.fac=="AML"]))) title("Box and wiskers plot for oncogenes of AML patients ") par(mfrow=c(1,1)) 8. Descriptive statistics for the ALL gene expression values of the Golub et al. (1999) data. (a) The ranges indicate strong diﬀerence in means. The range of the mean and of the median are similar. The bulk of the data seems symmetric. > range(apply(golub[,gol.fac=="ALL"],1,mean)) [1] -1.330984 3.278551 > range(apply(golub[,gol.fac=="ALL"],1,median)) [1] -1.36832 3.35455 (b) The range of the standard deviation is somewhat smaller than of the re-scaled IQR and MAD. > range(apply(golub[,gol.fac=="ALL"],1,sd)) [1] 0.1336206 2.0381309 > range(apply(golub[,gol.fac=="ALL"],1,function(x) IQR(x)/1.349)) [1] 0.1153113 2.7769867 > range(apply(golub[,gol.fac=="ALL"],1,mad)) 226 APPENDIX A. ANSWERS TO EXERCISES [1] 0.1056649 2.9656744 Answers to exercises of Chapter 3: Important Distributions 1. Binomial (a) P(X = 24) = 0.1046692, P(X ≤ 24) = 0.5557756, and P(X ≥ 30) = 0.0746237. (b) P(20 ≤ X ≤ 30) = 0.83856, P(20 ≤ X) = 0.8830403. (c) P(20 ≤ XorX ≥ 40) = 0.8830403, and P(20 ≤ XandX ≥ 10) = 0.999975. (d) E(X) = 24, var(X) = 3.794733 Use: sqrt(60 * 0.4 *0.6) (e) x 0.025 = 17, x 0.5 = 24, and x 0.975 = 32. 2. Standard Normal. (a) P(1.6 < Z < 2.3) = 0.04408. (b) P(Z < 1.64) = 0.9495. (c) P(−1.64 < Z < −1.02) = 0.1034. (d) P(0 < Z < 1.96) = 0.4750. (e) P(−1.96 < Z < 1.96) = 0.9500. (f) z 0.025 = −1.959964, z 0.05 = −1.644854, z 0.5 = 0, z 0.95 = 1.644854, and z 0.975 = 1.959964. 3. Normal. (a) P(X < 12) = 0.8413. (b) P(X > 8) = 0.8413. (c) P(9 < X < 10, 5) = 0.2917. (d) The quantiles x 0.025 = 6.080072, x 0.5 = 10, and x 0.975 = 13.91993. 4. T-distribution. (a) P(T 6 < 1) = 0.8220412. (b) P(T 6 > 2) = 0.04621316. (c) P(−1 < T 6 < 1) = 0.6440823. 227 (d) P(−2 < T 6 < −2) = 0.9075737. (e) t 0.025 = −2.446912, t 0.5 = 0, and t 0.975 = 2.446912. 5. F distribution. (a) P(F 8,5 < 3) = 0.8792198. (b) P(F 8,5 > 4) = 0.07169537. (c) P(1 < F 8,5 < 6) = 0.4931282. (d) The quantiles f 0.025 = 0.2075862, f 0.5 = 1.054510, and f 0.975 = 6.757172. 6. Chi-squared distribution. (a) P(χ 2 10 < 3) = 0.01857594. (b) P(χ 2 10 > 4) = 0.947347. (c) P(1 < χ 2 10 < 6) = 0.1845646. (d) The quantiles g 0.025 = 3.246973, g 0.5 = 9.341818, and g 0.975 = 20.48318. 7. MicroRNA. (a) P(X = 14) = dbinom(14, 20, 0.7) = 0.191639. (b) P(X ≤ 14) = pbinom(14, 20, 0.7) = 0.5836292. (c) P(X > 10) = 1 − P(X ≤ 10) = 1 −pbinom(10, 20, 0.7) = 0.9520381. (d) P(10 ≤ X ≤ 15) = P(X ≤ 15)−P(X ≤ 9) = pbinom(15, 20, 0.7) −pbinom(9, 20, 0.7) = 0.7453474. (e) 20 · 0.7 = 14. (f) sqrt(20* 0.7 * 0.3)=2.04939. 8. Zyxin. (a) P(X ≤ 1.2) =pnorm(1.2,1.6,0.4)=0.1586553. (b) P(1.2 ≤ X ≤ 2.0) =pnorm(2.0,1.6,0.4) - pnorm(1.2,1.6,0.4)=0.6826895. (c) P(2.4 ≤ X ≤ 0.8) =pnorm(2.4,1.6,0.4) - pnorm(0.8,1.6,0.4)=0.9544997. 228 APPENDIX A. ANSWERS TO EXERCISES (d) x 0.025 =qnorm(0.025,1.6,0.4)=0.8160144. Similarly, x 0.975 = 2.383986. (e) x <- rnorm(1000,1.6,0.4) gives mean(x) = 1.608401 and sd(x)=0.4022082. Both are close to the values in the population. 9. Some computations on Golub et al. (1999) data. (a) The tree larges t-value 57.8, 55.2, and 47.5 are extremely large. data(golub, package="multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) tval <- apply(golub[,gol.fac=="ALL"],1,function(x) sqrt(27) * mean(x)/sd(x)) o <- order(tval,decreasing=TRUE) tval[o[1:3]] golub.gnames[o[1:3],2] (b) The scrip below gives 2185 ratios between 0.5 and 1.5. sdall <- apply(golub[,gol.fac=="ALL"],1, sd) sdaml <- apply(golub[,gol.fac=="AML"],1, sd) sdratio <- sdall/sdaml sum( sdratio > 0.5 & sdratio < 1.5) 10. Extreme value investigation. The blue line (extreme value) ﬁts to the black line (density of generated extreme data) much better than the red line (normal distribution). an <- sqrt(2*log(n)) - 0.5*(log(log(n))+log(4*pi))*(2*log(n))^(-1/2) bn <- (2*log(n))^(-1/2) e <- double(); n <- 10000 # Serfling p.90 for (i in 1:1000) e[i] <- (max(rnorm(n))-an)/bn plot(density(e),ylim=c(0,0.5)) f<-function(x){exp(-x)*exp(-exp(-x))} curve(f,range(density(e)$x),add=TRUE,col = "blue")
Answers exercise chapter 4: Estimation and Inference
1. Gene CD33. Use agrep("^CD33",golub.gnames[,2]) to ﬁnd 808.
(a) The code
229
library(multtest);data(golub)
gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
shapiro.test(golub[i,gol.fac=="ALL"])
gives p-value = 0.592 and changing ALL into AML gives p-value
= 0.2583. Hence, for normality is accepted.
(b) var.test(golub[i,] ~ gol.fac) gives p-value = 0.1095 so equal-
ity of variances is accepted.
(c) t.test(golub[i,] ~ gol.fac, var.equal = TRUE) gives p-value = 1.773e-09,
so equality of means is rejected.
(d) Yes, t = -7.9813 is quite extreme.
2. Gene MYBL2 V-myb avian myeloblastosis viral oncogene homolog-like
2. Take i <- 1788.
(a) Use boxplot(golub[i,] ~ gol.fac) to observe from the box-
plot that one is quite certain that the null-hypothesis of no exper-
imental eﬀect holds.
(b) t.test(golub[i,] ~ gol.fac, var.equal = TRUE) gives p-value = 0.8597,
so that the null hypothesis of equal means is accepted.
3. HOXA9. Use i <- 1391.
(a) shapiro.test(golub[i,gol.fac=="ALL"]) gives p-value = 1.318e-07,
so that normality is rejected.
(b) wilcox.test(golub[i,] ~ gol.fac) gives p-value = 7.923e-05,
so that equality of means is rejected. Note that the p-value from
Grubbs test of the ALL expression values is 0.00519, so the null
hypothesis of no outliers is rejected. Nevertheless the Welch two-
sample T-test is also rejects the null-hypothesis of equal means.
Its t-value equals -4.3026 and is quite large.
4. Zyxin.
(a) Searching NCBI UniGene on zyxin gives BC002323.2.
(b) Use chisq.test(as.data.frame(table(read.GenBank(c("BC002323.2"))))$Freq) to ﬁnd p-value < 2.2e-16, so that the null-hypothesis of equal frequencies is rejected. 230 APPENDIX A. ANSWERS TO EXERCISES (c) We download and store the frequencies of the sequences in x and y. Next the empirical probabilities from y are use to predict the frequencies from y. x <- as.data.frame(table(read.GenBank(c("X94991.1"))))$Freq
y <- as.data.frame(table(read.GenBank(c("BC002323.2"))))$Freq >chisq.test(x, p=y/sum(y)) Chi-squared test for given probabilities data: x X-squared = 0.0277, df = 3, p-value = 0.9988 5. Gene selection. ptg <- apply(golub, 1, function(x) t.test(x ~ gol.fac, alternative = c("greater"))$p.value)
golub.gnames[order(ptg)[1:10],2]
6. Antigenes.
library(multtest); data(golub)
gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
pt <- apply(golub, 1, function(x) t.test(x ~ gol.fac)$p.value) index <-agrep("^antigen",golub.gnames[,2]) golub.index<-golub[index,] pt.index<-pt[index] golub.gnames.index<-golub.gnames[index,] golub.gnames.index[order(pt.index)[1:length(index)],2] 7. Genetic Model. From the output below the null hypothesis that the probabilities are as speciﬁed is accepted. > chisq.test(x=c(930,330,290,90),p=c(9/16,3/16,3/16,1/16)) Chi-squared test for given probabilities data: c(930, 330, 290, 90) X-squared = 4.2276, df = 3, p-value = 0.2379 8. Comparing two genes. 231 all66 <- golub[66,gol.fac=="ALL"] all790 <- golub[790,gol.fac=="ALL"] boxplot(all66,all790) mean(all66);mean(all790) median(all66);median(all790) sd(all66);sd(all790) IQR(all66)/1.349 ;IQR(all790)/1.349 mean(all66);mean(all790) mad(all66);mad(all790) shapiro.test(all66);shapiro.test(all790) 9. Normality tests. library(multtest);data(golub) gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) allsh <- apply(golub[,gol.fac=="ALL"], 1, function(x) shapiro.test(x)$p.value)
amlsh <- apply(golub[,gol.fac=="AML"], 1, function(x) shapiro.test(x)$p.value) > 100 * sum(allsh>0.05)/length(allsh) [1] 58.27598 > 100 * sum(amlsh>0.05)/length(amlsh) [1] 78.5644 > 100 * sum(allsh>0.05 & allsh>0.05)/length(allsh) [1] 58.27598 10. Two-sample tests on gene expression values of the Golub et al. (1999) data. (a) data(golub, package = "multtest"); gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) pt <- apply(golub, 1, function(x) t.test(x ~ gol.fac)$p.value)
pw <- apply(golub, 1, function(x) wilcox.test(x ~ gol.fac)$p.value) o <- order(pt,decreasing=FALSE) > golub.gnames[o[1:10],2] [1] "Zyxin" [2] "FAH Fumarylacetoacetate" [3] "APLP2 Amyloid beta (A4) precursor-like protein 2" [4] "LYN V-yes-1 Yamaguchi sarcoma viral related oncogene homolog" [5] "CST3 Cystatin C (amyloid angiopathy and cerebral hemorrhage)" 232 APPENDIX A. ANSWERS TO EXERCISES [6] "X-LINKED HELICASE II" [7] "RB1 Retinoblastoma 1 (including osteosarcoma)" [8] "TOP2B Topoisomerase (DNA) II beta (180kD)" [9] "TCRA T cell receptor alpha-chain" [10] "T-COMPLEX PROTEIN 1, GAMMA SUBUNIT" (b) > o <- order(pw,decreasing=FALSE) > golub.gnames[o[1:10],2] [1] "FAH Fumarylacetoacetate" [2] "Zyxin" [3] "CST3 Cystatin C (amyloid angiopathy and cerebral hemorrhage)" [4] "ELA2 Elastatse 2, neutrophil" [5] "TCF3 Transcription factor 3 (E2A immunoglobulin enhancer binding factors E12/E47)" [6] "Macmarcks" [7] "LYN V-yes-1 Yamaguchi sarcoma viral related oncogene homolog" [8] "CD33 CD33 antigen (differentiation antigen)" [9] "VIL2 Villin 2 (ezrin)" [10] "APLP2 Amyloid beta (A4) precursor-like protein 2" 11. Biological hypotheses. (a) n = 1000, p = 0.05 so np = 50 (b) pbinom(9,1000,.05)=5.24 · 10 −13 . (c) sum(dbinom(6:1000,1000,.05))=1. (d) sum(dbinom(2:8,1000,.05))= 8.8 · 10 −14 . 12. Programming some tests. (a) data(golub,package="multtest") gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) x <- golub[1042,gol.fac=="ALL"] n <- length(x) y <- golub[1042,gol.fac=="AML"] m <- length(y) t <- (mean(x)-mean(y))/sqrt(var(x)/n + var(y)/m) v <- (var(x)/n + var(y)/m)^2/( (var(x)/n)^2/(n-1) + (var(y)/m)^2/(m-1) ) 2*pt(-abs(t),v) mean(x) - mean(y) + qt(0.025,v)* sqrt(var(x)/n + var(y)/m) mean(x) - mean(y) + qt(0.975,v)* sqrt(var(x)/n + var(y)/m) 233 (b) z <- golub[1042,] > sum(rank(z)[1:27]) - 0.5*27*(27+1) [1] 284 (c) x <- golub[1042,gol.fac=="ALL"] y <- golub[1042,gol.fac=="AML"] w <- 0 for (i in 1:27) w <- w + sum(x[i]>y) > w [1] 284 Answers to exercises of Chapter 5 Linear Models 1. Analysis of gene expressions of B-cell ALL patients. library(ALL); data(ALL) ALLB <- ALL[,ALL$BT %in% c("B","B1","B2","B3","B4")]
> table(ALLB$BT) B B1 B2 B3 B4 T T1 T2 T3 T4 5 19 36 23 12 0 0 0 0 0 psw <- apply(exprs(ALLB), 1, function(x) shapiro.test(residuals(lm(x ~ ALLB$BT)))$p.value) library(lmtest) pbp <-apply(exprs(ALLB), 1, function(x) as.numeric(bptest(lm(x ~ ALLB$BT),studentize = FALSE)$p.value)) > sum(psw > 0.05) [1] 6847 > sum(pbp > 0.05) [1] 10057 > sum(psw > 0.05 & pbp > 0.05) [1] 6262 2. Further analysis of gene expressions of B-cell ALL patients. > panova <- apply(exprs(ALLB), 1, function(x) anova(lm(x ~ ALLB$BT))$Pr[1]) > featureNames(ALLB)[panova<0.000001] [1] "1125_s_at" "1126_s_at" "1134_at" "1389_at" "1500_at" 234 APPENDIX A. ANSWERS TO EXERCISES [6] "1866_g_at" "1914_at" "205_g_at" "31472_s_at" "31615_i_at" [11] "31616_r_at" "33358_at" "35614_at" "35991_at" "36873_at" [16] "37809_at" "37902_at" "38032_at" "38555_at" "39716_at" [21] "40155_at" "40268_at" "40493_at" "40661_at" "40763_at" [26] "41071_at" "41139_at" "41448_at" "873_at" > pkw <- apply(exprs(ALLB), 1, function(x) kruskal.test(x ~ ALLB$BT)$p.value) > featureNames(ALLB)[pkw<0.000001] [1] "1389_at" "1866_g_at" "38555_at" "40155_at" "40268_at" > panovasmall <- panova < 0.001 > pkwsmall <- pkw < 0.001 > table(panovasmall,pkwsmall) pkwsmall panovasmall FALSE TRUE FALSE 12172 38 TRUE 124 291 There are 124 signiﬁcant gene expressions from ANOVA which are not signiﬁcant on Kruskal-Wallis. There are only 38 signiﬁcant gene ex- pressions from Kruskal-Wallis which are non-signiﬁcant according to ANOVA. The tests agree on the majority of gene expressions. 3. Finding the ten best best genes among gene expressions of B-cell ALL patients. > sort(panova)[1:10] 1914_at 1389_at 38555_at 33358_at 40268_at 39716_at 1.466523e-14 5.891702e-14 4.873245e-10 1.117406e-09 1.145502e-09 4.748615e-09 40763_at 37809_at 36873_at 1866_g_at 5.256410e-09 2.155457e-08 2.402379e-08 3.997065e-08 > sort(pkw)[1:10] 1389_at 40268_at 38555_at 1866_g_at 40155_at 1914_at 2.348192e-09 7.764046e-08 1.123068e-07 2.335279e-07 6.595926e-07 1.074525e-06 1125_s_at 40662_g_at 38032_at 40661_at 1.346907e-06 1.384281e-06 1.475170e-06 1.719456e-06 npanova <- names(sort(panova)[1:10]) npkw <- names(sort(pkw)[1:10]) 235 > intersect(npanova,npkw) [1] "1914_at" "1389_at" "38555_at" "40268_at" "1866_g_at" 4. A simulation study for ANOVA. > x <- matrix(rnorm(90000),nrow = 10000, ncol = 9) > a <- gl(3,3) > panova <- apply(x, 1, function(x) anova(lm(x ~ a))$Pr[1])
> sum(panova<0.05)
[1] 514
The number of false positives is 514. The expected number is α · n =
0.05 · 10, 000 = 500, which is quite close to the observed.
A matrix with diﬀerences between three groups of gene expression val-
ues.
sigma <- 1; n <- 10000
data <- cbind(matrix(rnorm(n*3,0,sigma),ncol=3),
matrix(rnorm(n*3,1,sigma), ncol = 3),matrix(rnorm(n*3,2,sigma), ncol = 3))
a <- gl(3,3)
panova <- apply(data, 1, function(x) anova(lm(x ~ a))$Pr[1]) > sum(panova<0.05) [1] 3757 > pkw <- apply(data, 1, function(x) kruskal.test(x ~ a)$p.value)
> sum(pkw<0.05)
[1] 1143
Thus the number of true positives from ANOVA is 3757 and the num-
ber of false negatives is 6243. For the Kruskal-Wallis test there are
1143 true positives and 8857 false negatives. This can be impoved by
increasing the number of gene expressions per group.
Answers to exercises of Chapter 6: Micro Array Analysis.
1. Gene ﬁltering on normality per group of B-cell ALL patients.
236 APPENDIX A. ANSWERS TO EXERCISES
library("genefilter")
data(ALL, package = "ALL")
ALLB <- ALL[,ALL$BT %in% c("B1","B2","B3","B4")] f1 <- function(x) (shapiro.test(x)$p.value > 0.05)
sel1 <- genefilter(exprs(ALL[,ALLB$BT=="B1"]), filterfun(f1)) sel2 <- genefilter(exprs(ALL[,ALLB$BT=="B2"]), filterfun(f1))
sel3 <- genefilter(exprs(ALL[,ALLB$BT=="B3"]), filterfun(f1)) sel4 <- genefilter(exprs(ALL[,ALLB$BT=="B4"]), filterfun(f1))
selected <- sel1 & sel2 & sel3 & sel4
library(limma)
x <- matrix(as.integer(c(sel2,sel3,sel4)),ncol = 3,byrow=FALSE)
colnames(x) <- c("sel2","sel3","sel4")
vc <- vennCounts(x, include="both")
vennDiagram(vc)
137 pass filter 2 but not the other
510 pass filter 2 and 3 but not 4
1019 pas filter 2 and 4 but not 3
5598 pass filter 2, 3 and 4. etc.
2. Analysis of gene expressions of B-cell ALL patients using Limma.
library("ALL"); library("limma");library("annaffy");library(hgu95av2.db)
data(ALL)
ALLB <- ALL[,ALL$BT %in% c("B1","B2","B3","B4")] design.ma <- model.matrix(~0 + factor(ALLB$BT))
colnames(design.ma) <- c("B1","B2","B3","B4")
cont.ma <- makeContrasts(B2-B1,B3-B2,B4-B3,levels=factor(ALLB$BT)) fit <- lmFit(ALLB, design.ma) fit1 <- contrasts.fit(fit, cont.ma) fit1 <- eBayes(fit1) topTable(fit1, coef=2,5,adjust.method="fdr") tab <- topTable(fit1, coef=2, number=20, adjust.method="fdr") anntable <- aafTableAnn(as.character(tab$ID), "hgu95av2", aaf.handler())
saveHTML(anntable, "ALLB1234.html", title = "B-cell ALL of stage 1,2,3,4")
3. Finding a row number: grep("1389_at",row.names(exprs(ALL))).
4. Remission (genezing) from acute lymphocytic leukemia (ALL).
237
library(ALL); data(ALL)
table(pData(ALL)$remission) remis <- which(pData(ALL)$remission %in% c("CR","REF"))
ALLrem <- ALL[,remis]
remfac <-factor(pData(ALLrem)$remission) pano <- apply(exprs(ALLrem),1,function(x) t.test(x ~ remfac)$p.value)
sum(pano<0.001)
> sum(pano<0.001)
[1] 45
library(hgu95av2.db)
names <- featureNames(ALLrem)[pano<.001]
ALLremsel<- ALLrem[names,]
symb <- mget(names, env = hgu95av2SYMBOL)
genenames <- mget(names,hgu95av2GENENAME)
listofgenenames <- as.list(hgu95av2GENENAME)
unlistednames <- unlist(listofgenenames[names],use.names=F)
> grep("p53",unlistednames)
[1] 12 21
> length(unique(unlistednames))
[1] 36
5. Remission achieved.
library(ALL); data(ALL)
ALLCRREF <- ALL[,which(ALL$CR %in% c("CR","REF"))] pano <- apply(exprs(ALLCRREF),1,function(x) t.test(x ~ ALLCRREF$CR)$p.value) > sum(pano<0.0001) [1] 11 > featureNames(ALLCRREF)[pano<.0001] [1] "1472_g_at" "1473_s_at" "1475_s_at" "1863_s_at" "34098_f_at" "36574_at" "38124_at" "38279_at" "41337_at" "577_at" "953_g_at" library("hgu95av2.db") affynames <- featureNames(ALLCRREF)[pano<.0001] genenames <- mget(affynames, env = hgu95av2GENENAME) 238 APPENDIX A. ANSWERS TO EXERCISES > grep("oncogene",genenames) [1] 1 2 3 affytot <- unique(featureNames(ALLCRREF)) genenamestot <- mget(affytot, env = hgu95av2GENENAME) > length(grep("oncogene",genenamestot)) [1] 239 > length(genenamestot) [1] 12625 > dat <- matrix(c(12625,239,11,3),2,byrow=TRUE) > fisher.test(dat) Fisher’s Exact Test for Count Data data: dat p-value = 0.002047 alternative hypothesis: true odds ratio is not equal to 1 95 percent confidence interval: 2.562237 54.915642 sample estimates: odds ratio 14.39959 6. Gene ﬁltering of ALL data. library("ALL") data("ALL") table(ALL$BT)
ALLT23 <- ALL[,which(ALL$BT %in% c("T2","T3"))] library(genefilter) f1 <- function(x) (shapiro.test(x)$p.value > 0.05)
f2 <- function(x) (t.test(x ~ ALLT23$BT)$p.value < 0.05)
sel1 <- genefilter(exprs(ALLT23[,ALLT23$BT=="T2"]), filterfun(f1)) sel2 <- genefilter(exprs(ALLT23[,ALLT23$BT=="T3"]), filterfun(f1))
sel3 <- genefilter(exprs(ALLT23), filterfun(f2))
> sum(sel1 & sel2 & sel3)
239
[1] 905
> sum(sel1 & sel2)
[1] 9388
> sum(sel3)
[1] 1204
7. Stages of B-cell ALL in the ALL data.
library("ALL")
library("limma");
allB <- ALL[,which(ALL$BT %in% c("B1","B2","B3","B4"))] facB123 <- factor(allB$BT)
cont.ma <- makeContrasts(B2-B1,B3-B2,B4-B3, levels=facB123)
design.ma <- model.matrix(~ 0 + facB123)
colnames(design.ma) <- c("B1","B2","B3","B4")
fit <- lmFit(allB, design.ma)
fit1 <- contrasts.fit(fit, cont.ma)
fit1 <- eBayes(fit1)
ID logFC AveExpr t P.Value adj.P.Val B
6048 35991_at 0.5964481 4.144598 6.624128 2.578836e-09 0.0000325578 10.842989
3909 33873_at 0.5707770 7.217570 6.083524 2.891823e-08 0.0001825464 8.625253
5668 35614_at 1.7248509 5.663477 5.961231 4.946078e-08 0.0002081474 8.132884
6776 36711_at -2.3664712 7.576108 -5.759565 1.187487e-07 0.0003054110 7.329631
7978 37902_at 0.8470235 4.258491 5.742783 1.276579e-07 0.0003054110 7.263298
> sum(fit1$p.value<0.05) [1] 4328 8. Analysis of public micro array data. library(GEOquery); library(limma); library(hgu95av2.db);library(annaffy) gds486 <- getGEO("GDS486"); eset486 <- GDS2eSet(gds486,do.log2=T) nrmissing <- apply(exprs(eset486), 1, function(x) sum(is.na(x)) ) eset486sel <- eset486[nrmissing<1,] pval486sel <- apply(exprs(eset486sel), 1, function(x) t.test(x ~ eset486sel$cell.line)$p.value) pval486 <- nrmissing pval486[pval486==0]<-pval486sel 240 APPENDIX A. ANSWERS TO EXERCISES pval486[pval486>1]<-1 gds711 <- getGEO("GDS711"); eset711 <- GDS2eSet(gds711,do.log2=T) nrmissing <- apply(exprs(eset711), 1, function(x) sum(is.na(x)) ) eset711sel <- eset711[nrmissing<1,] panova711sel <- apply(exprs(eset711sel), 1, function(x) anova(lm(x ~ eset711sel$disease.state))$Pr[1]) pval711sel <- panova711sel pval711 <- nrmissing pval711[pval711==0]<-pval711sel pval711[pval711>1]<-1 gds2126 <- getGEO("GDS2126"); eset2126 <- GDS2eSet(gds2126,do.log2=T) nrmissing <- apply(exprs(eset2126), 1, function(x) sum(is.na(x)) ) eset2126sel <- eset2126[nrmissing<1,] pval2126sel <- apply(exprs(eset2126sel), 1, function(x) anova(lm(x ~ eset2126sel$disease.state))$Pr[1]) pval2126 <- nrmissing pval2126[pval2126==0]<-pval2126sel pval2126[pval2126>1]<-1 sumpval <- pval486 + pval711 + pval2126 o <- order(sumpval,decreasing=FALSE) genenames <- names(sumpval[o[1:20]]) symb <- "aap" for (i in 1:20) symb[i] <- get(genenames[i], env = hgu95av2SYMBOL) > symb [1] "GADD45A" "DUSP4" "OAS1" "STAT1" "STAT1" "AKR1C3" "PSMB9" "OAS2" "STAT1" "BUB1B" "UBE2L6" "STAT1" "ZFP36L2" "IL1R1" "IL8" [16] "TKT" "NFKB1" "SLC7A5" "CXCL2" "DLG5" library("KEGG");library("GO");library("annaffy") atab <- aafTableAnn(genenames, "hgu95av2", aaf.handler() ) saveHTML(atab, file="ThreeExperiments.html") # p53 plays a role. 9. Analysis of genes from a GO search. library(ALL) data(ALL,package="ALL") 241 ALLP <- ALL[,ALL$mol.biol %in% c("ALL1/AF4","BCR/ABL","NEG")]
neg <- which(ALLP$mol.biol=="NEG") aal1 <- which(ALLP$mol.biol=="ALL1/AF4")
bcr <- which(ALLP$mol.biol=="BCR/ABL") orderpat <- c(neg,aal1,bcr) ALLP <- ALL[,ALL$mol.biol %in% c("ALL1/AF4","BCR/ABL","NEG")]
ALLPo <- ALLP[,c(neg,aal1,bcr)]
facnr <- c(rep(1,74),rep(2,10),rep(3,37))
nab.fac <- factor(facnr,levels=1:3, labels= c("NEG","ALL1/AF4","BCR/ABL"))
panova <- apply(exprs(ALLPo), 1, function(x) anova(lm(x ~ nab.fac))$Pr[1]) library("GO"); library("annotate"); library("hgu95av2") GOTerm2Tag <- function(term) { GTL <- eapply(GOTERM, function(x) {grep(term, x@Term, value=TRUE)}) Gl <- sapply(GTL, length) names(GTL[Gl>0]) } > GOTerm2Tag("protein-tyrosine kinase") [1] "GO:0004713" probes <- hgu95av2GO2ALLPROBES$"GO:0004713"
> sum(panova[probes]<0.05)
[1] 86
> sum(panova[probes]<1)
[1] 320
> sum(panova<0.05)
[1] 2581
> sum(panova<1)
[1] 12625
> fisher.test(matrix(c(12625, 2581,320,86),2,byrow=TRUE))
Fisher’s Exact Test for Count Data
242 APPENDIX A. ANSWERS TO EXERCISES
data: matrix(c(12625, 2581, 320, 86), 2, byrow = TRUE)
p-value = 0.03222
alternative hypothesis: true odds ratio is not equal to 1
95 percent confidence interval:
1.019848 1.679625
sample estimates:
odds ratio
1.314569
the odds ratio differs significantly from zero; there are more significant results among the probes related to protein-tyrosine kinase.
Answers to exercises of Chapter 7: Cluster Analysis and Trees.
1. Cluster analysis on the ”Zyxin” expression values of the Golub et al.
(1999) data.
data(golub, package="multtest")
data <- data.frame(golub[2124,])
gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML"))
stripchart(golub[2124,]~gol.fac, pch=as.numeric(gol.fac))
plot(hclust(dist(clusdata,method="euclidian"),method="single"))
initial <- as.matrix(tapply(golub[2124,],gol.fac,mean), nrow = 2, ncol=1, byrow=TRUE)
cl<- kmeans(data, initial, nstart = 10)
table(cl$cluster,gol.fac) n <- length(data); nboot<-1000 boot.cl <- matrix(0,nrow=nboot,ncol = 2) for (i in 1:nboot){ dat.star <- data[sample(1:n,replace=TRUE)] cl <- kmeans(dat.star, initial, nstart = 10) boot.cl[i,] <- c(cl$centers[1,],cl$centers[2,]) } > quantile(boot.cl[,1],c(0.025,0.975)) 2.5% 97.5% -1.07569310 -0.03344292 243 > quantile(boot.cl[,2],c(0.025,0.975)) 2.5% 97.5% 0.731493 1.784468 2. Close to CCND3 Cyclin D3. library("genefilter"); data(golub, package = "multtest") closeg <- genefinder(golub, 1042, 10, method = "euc", scale = "none") golub.gnames[closeg[[1]][[1]],2] boxplot(golub[394,] ~gol.fac) 3. MCM3. data(golub, package = "multtest") x <- golub[2289,]; y <- golub[2430,] plot(x,y) which.min(y) # the plot suggests the smallest y as the outlier > cor.test(x[-21],y[-21]) Pearson’s product-moment correlation data: x[-21] and y[-21] t = 10.6949, df = 35, p-value = 1.42e-12 alternative hypothesis: true correlation is not equal to 0 95 percent confidence interval: 0.7690824 0.9341905 # much smaller sample estimates: cor 0.875043 # much larger than 0.6376217 nboot <- 1000; boot.cor <- matrix(0,nrow=nboot,ncol = 1) data <- matrix(c(x[-21],y[-21]),ncol=2,byrow=FALSE) for (i in 1:nboot){ dat.star <- data[sample(1:nrow(data),replace=TRUE),] boot.cor[i,] <- cor(dat.star)[2,1]} > mean(boot.cor) [1] 0.8725835 # very similar to cor.test > quantile(boot.cor[,1],c(0.025,0.975)) 2.5% 97.5% 0.7755743 0.9324625 # very similar to cor.test 244 APPENDIX A. ANSWERS TO EXERCISES 4. Cluster analysis on part of Golub data. library(multtest);data(golub); gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) o1 <- grep("oncogene",golub.gnames[,2]) plot(hclust(dist(golub[o1,],method="euclidian"),method="single")) o2 <- grep("antigene",golub.gnames[,2]) plot(hclust(dist(golub[o2,],method="euclidian"),method="single")) o3 <- grep("receptor",golub.gnames[,2]) plot(hclust(dist(golub[o3,],method="euclidian"),method="single")) 5. Principal Components Analysis on part of the ALL data. library(ALL); data(ALL) ALLB <- ALL[,ALL$BT %in% c("B1","B2","B3")]
panova <- apply(exprs(ALLB), 1, function(x) anova(lm(x ~ ALLB$BT))$Pr[1])
ALLBsp <- ALLB[panova<0.001,]
> dim(exprs(ALLBsp))
[1] 499 78
> min(cor(exprs(ALLBsp)))
[1] 0.5805595
> eigen(cor(exprs(ALLBsp)))$values[1:5] [1] 65.2016203 2.9652965 2.4781567 0.7556439 0.6040647 data <- exprs(ALLBsp); p <- ncol(data); n <- nrow(data) ; nboot<-1000 eigenvalues <- array(dim=c(nboot,p)) for (i in 1:nboot){dat.star <- data[sample(1:n,replace=TRUE),] eigenvalues[i,] <- eigen(cor(dat.star))$values}
> for (j in 1:p) print(quantile(eigenvalues[,j],c(0.025,0.975)))
2.5% 97.5%
63.43550 66.77785
2.5% 97.5%
2.575413 3.530350
2.5% 97.5%
2.081573 2.889933
2.5% 97.5%
245
0.6475809 0.9942871 #Hence, the first three are significant!
2.5% 97.5%
0.5067404 0.7482680
2.5% 97.5%
biplot(princomp(data,cor=TRUE),pc.biplot=T,cex=0.5,expand=0.8)
6. Some correlation matrices.
eigen(matrix(c(1,-0.8,-0.8,1),nrow=2))
eigen(matrix(c(1,0.8,0.8,0.8,1,0.8,0.8,0.8,1),nrow=3))
eigen(matrix(c(1,-0.5,-0.5,-0.5,1,-0.5,-0.5,-0.5,1),nrow=3))
> 2.6/3 * 100
[1] 86.66667
> eigen(matrix(c(1,0.8,0.8,0.8,1,0.8,0.8,0.8,1),nrow=3))$vectors [,1] [,2] [,3] [1,] -0.5773503 0.8164966 0.0000000 [2,] -0.5773503 -0.4082483 -0.7071068 [3,] -0.5773503 -0.4082483 0.7071068 Answers to exercises of Chapter 8: Classiﬁcation Methods. 1. Classiﬁcation tree of Golub data. Use recursive partitioning in rpart library(multtest);data(golub); gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) maxgol <- apply(golub[,gol.fac=="ALL"], 1, function(x) max(x)) mingol <- apply(golub[,gol.fac=="AML"], 1, function(x) min(x)) sum(maxgol < mingol) > which.min(maxgol - mingol) [1] 2124 > golub.gnames[2124,] [1] "4847" "Zyxin" "X95735_at" > boxplot(golub[2124,] ~gol.fac) gol.rp <- rpart(gol.fac ~ golub[2124,], method="class", cp=0.001) plot(gol.rp, branch=0,margin=0.1); text(gol.rp, digits=3, use.n=TRUE) 246 APPENDIX A. ANSWERS TO EXERCISES grep("Gdf5",golub.gnames[,2]) > grep("Gdf5",golub.gnames[,2]) [1] 2058 gol.rp <- rpart(gol.fac ~ golub[2058,], method="class", cp=0.001) plot(gol.rp, branch=0,margin=0.1); text(gol.rp, digits=3, use.n=TRUE) gol.rp <- rpart(gol.fac ~., data.frame(t(golub)), method="class", cp=0.001) plot(gol.rp, branch=0,margin=0.1); text(gol.rp, digits=3, use.n=TRUE) 2. Sensitivity versus speciﬁcity. (a) library(multtest);library(ROCR);data(golub) golub.clchanged <- -golub.cl +1 pred <- prediction(golub[1042,], golub.clchanged) perf <- performance(pred, "sens", "spec") plot(perf) (b) The function is essentially the same. (c) Use auc as before. 3. Comparing Classiﬁcation Methods. library(rpart) predictors <- matrix(rnorm(100*4,0,1),100,4) colnames(predictors) <- letters[1:4] groups <- gl(2,50) simdata <- data.frame(groups,predictors) rp<-rpart(groups ~ a + b + c + d,method="class",data=simdata) predicted <- predict(rp,type="class") table(predicted,groups) plot(rp, branch=0,margin=0.1); text(rp, digits=3, use.n=TRUE) > table(predicted,groups) groups predicted 1 2 1 41 12 247 2 9 38 library(e1071) svmest <- svm(predictors, groups, data=df, type = "C-classification", kernel = "linear") svmpred <- predict(svmest, predictors, probability=TRUE) > table(svmpred, groups) groups svmpred 1 2 1 31 25 2 19 25 library(nnet) nnest <- nnet(groups ~ ., data = simdata, size = 5,maxit = 500, decay = 0.01, MaxNWts = 5000) pred <- predict(nnest, type = "class") > table(pred, groups) # prints confusion ma groups pred 1 2 1 45 10 2 5 40 The misclassiﬁcation rate of rpart, svm, and nnet is, respectively, 21/100, 44/100, and 15/100. If we increase the number of predictors, then the misclassiﬁcation rate decreases. 4. Prediction of achieved remission. library(ALL); library(hgu95av2.db); library(rpart); data(ALL) ALLrem <- ALL[,which(pData(ALL)$remission %in% c("CR","REF"))]
remfac <-factor(pData(ALLrem)$remission) pano <- apply(exprs(ALLrem),1,function(x) t.test(x ~ remfac)$p.value)
names <- featureNames(ALLrem)[pano<.001]
ALLremsel<- ALLrem[names,]
data <- data.frame(t(exprs(ALLremsel)))
all.rp <- rpart(remfac ~., data, method="class", cp=0.001)
plot(all.rp, branch=0,margin=0.1); text(all.rp, digits=3, use.n=TRUE)
rpart.pred <- predict(all.rp, type="class")
> table(rpart.pred,remfac)
remfac
248 APPENDIX A. ANSWERS TO EXERCISES
rpart.pred CR REF
CR 93 1
REF 6 14
> 7/(93+1+6+14)
[1] 0.06140351
> mget(c("1840_g_at","36769_at","1472_g_at","854_at"), env = hgu95av2GENENAME)
$‘1840_g_at‘ [1] NA$‘36769_at‘
[1] "retinoblastoma binding protein 5"
$‘1472_g_at‘ [1] "v-myb myeloblastosis viral oncogene homolog (avian)"$‘854_at‘
[1] "B lymphoid tyrosine kinase"
5. Gene selection by area under the curve.
library(ROCR); data(golub, package = "multtest")
gol.true <- factor(golub.cl,levels=0:1,labels= c("TRUE","FALSE"))
auc.values <- apply(golub,1,
function(x) performance(prediction(x, gol.true),"auc")@y.values[[1]])
o <- order(auc.values,decreasing=TRUE)
golub.gnames[o[1:25],2]
6. Classiﬁcation Tree for Ecoli.
colnames(ecoli) <- c("SequenceName","mcg","gvh","lip","chg","aac","alm1","alm2","ecclass")
ecolisel<- ecoli[which(ecoli$ecclass %in% c("cp","im","pp")),] ecolisel$ecclass <- factor(ecolisel$ecclass, levels=c("cp","im","pp")) library(rpart) rpfit <- rpart(ecolisel$ecclass ~ mcg + gvh + lip + aac + alm1 + alm2,data=ecolisel, method="class")
plot(rpfit, branch=1,margin=0.1); text(rpfit, digits=3, use.n=TRUE)
title(main = "rpartfit ecoli classes cp im and pp")
249
predictedclass <- predict(rpfit, type="class")
table(predictedclass,ecolisel$ecclass) #predictors are alm1, gvh and im > (1+2+7+4)/length(ecolisel$ecclass)
[1] 0.05166052
Answers to exercises of Chapter 9: Analyzing Sequences
1. Writing to a FASTA ﬁle.
choosebank("genbank"); library(seqinr)
query("ccnd3hs","sp=homo sapiens AND k=ccnd3@")
ccnd3 <- sapply(ccnd3hs$req, getSequence) x1 <- DNAStringSet(c2s(ccnd3[[1]])) write.XStringSet(x1, file="ccnd3.fa", format="fasta", width=80) ccnd3c2sn <- sapply(ccnd3, c2s) x1 <- DNAStringSet(ccnd3c2sn) write.XStringSet(x1, file="ccnd3n.fa", format="fasta", width=80) An alternative would be to use the write.dna function of the ape package. 2. Dotplot of sequences. seq1 <- sample(c("A","G","C","T"),100,rep=TRUE,prob=c(0.1,0.4,0.4,0.1)) seq2 <- sample(c("A","G","C","T"),100,rep=TRUE,prob=c(0.1,0.4,0.4,0.1)) par(mfrow=c(1,2)) dotPlot(seq1, seq2, main = "Dot plot of different random sequences\nwsize = 1, wstep = 1, nmatch = 1") dotPlot(seq1, seq1, main = "Dot plot of equal random sequnces\nwsize = 1, wstep = 1, nmatch = 1") par(mfrow=c(1,1)) par(mfrow=c(1,2)) dotPlot(seq1, seq2, main = "Dot plot of different random sequences\nwsize = 3, wstep = 3, nmatch = 3") dotPlot(seq1, seq1, main = "Dot plot of equal random sequnces\nwsize = 3, wstep = 3, nmatch = 3") par(mfrow=c(1,1)) par(mfrow=c(1,2)) dotPlot(seq1, seq1, main = "Dot plot of different random sequences\nwsize = 3, wstep = 3, nmatch = 3") 250 APPENDIX A. ANSWERS TO EXERCISES dotPlot(seq1, seq1[100:1], main = "Dot plot of equal random sequnces\nwsize = 3, wstep = 3, nmatch = 3") par(mfrow=c(1,1)) x <- c("RPLWVAPDGHIFLEAFSPVYK") y <- c("RPLWVAPDGHIFLEAFSPVYK") z <- c("PLWISPSDGRIILESFSPLAE") choosebank("genbank"); library(seqinr) query("ccnd3hs","sp=homo sapiens AND k=ccnd3@") ccnd3 <- sapply(ccnd3hs$req, getSequence)
sapply(ccnd3hs$req, getName) ccnd3prot <- sapply(ccnd3hs$req, getTrans)
dotPlot(ccnd3prot[[1]], s2c("EEEVFPLAMN"), main = "Dot plot of two protein\nwsize = 1, wstep = 1, nmatch = 1")
dotPlot(ccnd3prot[[7]], ccnd3prot[[8]], main = "Dot plot of two protein\nwsize = 1, wstep = 1, nmatch = 1")
dotPlot(s2c(x), s2c(z), main = "Dot plot of two protein\nwsize = 1, wstep = 1, nmatch = 1")
3. Local alignment.
library(seqinr);library(Biostrings);data(BLOSUM50)
x <- s2c("HEAGAWGHEE"); y <- s2c("PAWHEAE")
s <- BLOSUM50[y,x]; d <- 8
F <- matrix(data=NA,nrow=(length(y)+1),ncol=(length(x)+1))
F[1,] <- 0 ; F[,1] <- 0
rownames(F) <- c("",y); colnames(F) <- c("",x)
for (i in 2:(nrow(F)))
for (j in 2:(ncol(F)))
{F[i,j] <- max(c(0,F[i-1,j-1]+s[i-1,j-1],F[i-1,j]-d,F[i,j-1]-d))}
> max(F)
[1] 28
4. Probability of more extreme alignment score.
library(seqinr);library(Biostrings);data(BLOSUM50)
randallscore <- c(1,1)
for (i in 1:1000) {
x <- c2s(sample(rownames(BLOSUM50),7, replace=TRUE))
y <- c2s(sample(rownames(BLOSUM50),10, replace=TRUE))
randallscore[i] <- pairwiseAlignment(AAString(x), AAString(y), substitutionMatrix = "BLOSUM50",
251
gapOpening = 0, gapExtension = -8, scoreOnly = TRUE)
}
> sum(randallscore>1)/1000
[1] 0.003
> plot(density(randallscore))
5. Prochlorococcus marinus.
library(seqinr)
choosebank("genbank")
query("ccmp","AC=AE017126 OR AC=BX548174 OR AC=BX548175")
ccmpseq <- sapply(ccmp$req,getSequence) gc <- sapply(ccmpseq, GC) > wilcox.test(gc[1:2],gc[3:9]) Wilcoxon rank sum test data: gc[1:2] and gc[3:9] W = 0, p-value = 0.05556 alternative hypothesis: true location shift is not equal to 0 > t.test(gc[1:2],gc[3:9]) Welch Two Sample t-test data: gc[1:2] and gc[3:9] t = -5.8793, df = 1.138, p-value = 0.08649 alternative hypothesis: true difference in means is not equal to 0 95 percent confidence interval: -0.4507417 0.1079848 sample estimates: mean of x mean of y 0.3362065 0.5075849 gc in the left group is lower, the tests are not significant. 6. Sequence equality. \begin{verbatim} library(seqinr) choosebank("genbank") query("ccnd3hs","sp=homo sapiens AND k=ccnd3@") 252 APPENDIX A. ANSWERS TO EXERCISES sapply(ccnd3hs$req,getLength)
> ccnd3prot <- sapply(ccnd3hs$req, getTrans) > table(ccnd3prot[[1]]) * A C D E F G H I K L M N P Q R S T V W Y 1 31 12 12 21 6 14 7 10 10 41 9 1 17 16 22 19 18 15 3 8 > table(ccnd3prot[[2]]) * A C D E F G H I K L M N P Q R S T V W Y 1 30 12 12 21 6 14 7 10 10 41 9 1 17 16 22 20 18 15 3 8 # Hence, there is only one difference! > which(!ccnd3prot[[1]]==ccnd3prot[[2]]) [1] 259 7. Conserved region. ID XRODRMPGMNTB; BLOCK AC PR00851A; distance from previous block=(52,131) DE Xeroderma pigmentosum group B protein signature BL adapted; width=21; seqs=8; 99.5%=985; strength=1287 XPB_HUMAN|P19447 ( 74) RPLWVAPDGHIFLEAFSPVYK 54 XPB_MOUSE|P49135 ( 74) RPLWVAPDGHIFLEAFSPVYK 54 P91579 ( 80) RPLYLAPDGHIFLESFSPVYK 67 XPB_DROME|Q02870 ( 84) RPLWVAPNGHVFLESFSPVYK 79 RA25_YEAST|Q00578 ( 131) PLWISPSDGRIILESFSPLAE 100 Q38861 ( 52) RPLWACADGRIFLETFSPLYK 71 O13768 ( 90) PLWINPIDGRIILEAFSPLAE 100 O00835 ( 79) RPIWVCPDGHIFLETFSAIYK 86 library(Biostrings);data(BLOSUM50) x <- c("RPLWVAPDGHIFLEAFSPVYK") y <- c("RPLWVAPDGHIFLEAFSPVYK") z <- c("PLWISPSDGRIILESFSPLAE") x == y pairwiseAlignment(AAString(x), AAString(z), substitutionMatrix = "BLOSUM50",gapOpening = 0, gapExtension = -8, scoreOnly = FALSE) > pairwiseAlignment(AAString(x), AAString(y), substitutionMatrix = "BLOSUM50",gapOpening = 0, gapExtension = -8, scoreOnly = FALSE) Global Pairwise Alignment 253 1: RPLWVAPDGHIFLEAFSPVYK 2: RPLWVAPDGHIFLEAFSPVYK Score: 154 > > z <- c("PLWISPSDGRIILESFSPLAE") > > x == y [1] TRUE > pairwiseAlignment(AAString(x), AAString(z), substitutionMatrix = "BLOSUM50",gapOpening = 0, gapExtension = -8, scoreOnly = FALSE) Global Pairwise Alignment 1: RPLWVAP-DGHIFLEAFSPVYK 2: -PLWISPSDGRIILESFSPLAE Score: 85 8. Plot of CG proportion from Celegans. (a) Produce a plot of the CG proportion of the chromosome I of Cel- egans (Celegans.UCSC.ce2) along a window of 100 nucleotides. Take the ﬁrst 10,000 nucleotides. library(seqinr) source("http://bioconductor.org/biocLite.R") biocLite("BSgenome.Celegans.UCSC.ce2") library(BSgenome.Celegans.UCSC.ce2) GCperc <- double() for (i in 1:10000) GCperc[i] <- GC(s2c(as.character(Celegans$chrI[i:(i+100)])))
plot(GCperc,type="l")
(b) A binding sequence of the enzyme EcoRV is the subsequence
GATATC. How many exact matches has Chromosome I of Cel-
egans.
> subseq <- "gatatc"
> countPattern(subseq, Celegans$chrI, max.mismatch = 0) [1] 3276 > length(s2c(as.character(Celegans$chrI))) * (1/4)^6
[1] 3681.759
9. Plot of codon usage.
254 APPENDIX A. ANSWERS TO EXERCISES
data(ec999)
ec999.uco <- lapply(ec999, uco, index="eff")
df <- as.data.frame(lapply(ec999.uco, as.vector))
row.names(df) <- names(ec999.uco[[1]])
global <- rowSums(df)
title <- "Codon usage in 999 E. coli coding sequences"
dotchart.uco(global, main = title)
choosebank("genbank"); library(seqinr)
query("ccndhs","sp=homo sapiens AND k=ccnd@")
ccnd <- sapply(ccndhs$req, getSequence) ccnd.uco <- lapply(ccnd3, uco, index="eff") df <- as.data.frame(lapply(ccnd.uco, as.vector)) row.names(df) <- names(ccnd.uco[[1]]) global <- rowSums(df) title <- "Codon usage in ccnd3 homo sapiens coding sequences" dotchart.uco(global, main = title) Answers to exercises of Chapter 10: Markov Models. 1. Visualize by a transition graph the following transition matrices. Con- sult your teacher. 2. Computing probabilities. The answers are provided by the following. > P <- matrix(c(3/4,1/4,1/2,1/2),2,2,byrow=T) > pi0 <- c(1/2,1/2) > pi0 %*% P [,1] [,2] [1,] 0.625 0.375 > P %*% P [,1] [,2] [1,] 0.6875 0.3125 [2,] 0.6250 0.3750 > P [,1] [,2] [1,] 0.75 0.25 [2,] 0.50 0.50 255 3. Programming GTR. Use π A = 0.15, π G = 0.35, π C = 0.35, π T = 0.15, α = 4, β = 0.5, γ = 0.4, δ = 0.3, = 0.2, and ζ = 4. (a) Program the rate matrix in such a manner that it is simple to adapt for other values of the parameters. library(Matrix) piA <- 0.15; piG <- 0.35; piC <- 0.35; piT <- 0.15 alpha <- 4; beta <- 0.5; gamma <- 0.4; delta <- 0.3 epsilon <- 0.2; zeta <- 4 Q <- matrix(data=NA,4,4) Q[1,2] <- alpha * piG; Q[1,3] <- beta * piC; Q[1,4] <- gamma * piT Q[2,1] <- alpha * piA; Q[2,3] <- delta * piC; Q[2,4] <- epsilon * piT Q[3,1] <- beta * piA; Q[3,2] <- delta * piG; Q[3,4] <- delta* piT Q[4,1] <- gamma * piA; Q[4,2] <- epsilon* piG; Q[4,3] <- zeta * piC diag(Q) <- 0 diag(Q) <- -apply(Q,1,sum) Q <- Matrix(Q) > Q 4 x 4 Matrix of class "dgeMatrix" [,1] [,2] [,3] [,4] [1,] -1.635 1.400 0.175 0.060 [2,] 0.600 -0.735 0.105 0.030 [3,] 0.075 0.105 -0.225 0.045 [4,] 0.060 0.070 1.400 -1.530 (b) The transversion rate is larger then the transition rate because the blocks outside the main diagonal have lower values. (c) The probability transition matrix is > P <- as.matrix(expm(Q)) > P [,1] [,2] [,3] [,4] [1,] 0.32199057 0.51569256 0.1392058 0.02311107 [2,] 0.22097363 0.64908639 0.1115233 0.01841667 256 APPENDIX A. ANSWERS TO EXERCISES [3,] 0.05203969 0.09913633 0.8263804 0.02244359 [4,] 0.04621015 0.08457814 0.6397090 0.22950271 rownames(P) <- colnames(P) <- StateSpace <- c("a","g","c","t") pi0 <- c(1/4,1/4,1/4,1/4) markov2 <- function(StateSpace,P,n){ seq <- matrix(0,nr=n,nc=1) seq[1] <- sample(StateSpace,1,replace=T,pi0) for(k in 1:(n-1)){ seq[k+1] <- sample(StateSpace,1,replace=T,P[seq[k],])} return(seq) } seq <- markov2(StateSpace,P,99) 4. Distance according to JC69. (a) accnr <- paste("AJ5345",26:27,sep="") seqbin <- read.GenBank(accnr, species.names = TRUE, as.character = FALSE) Down load the sequences AJ534526 and AJ534527. Hint: Use as.character = TRUE in the read.GenBank function. 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Statistical Applications in Genetics and Molecular Biology, 3, No. 1, Article 3. Smyth, G. K. (2005). Limma: linear models for microarray data. In: ’Bioin- formatics and Computational Biology Solutions using R and Biocon- ductor’. R. Gentleman, V. Carey, S. Dudoit, R. Irizarry, W. Huber (eds), Springer, New York, pages 397–420. Venables W.N. & Ripley B.D. (2000). S programming. New York: Springer. Venables, W. N. & Ripley, B. D. (2002) Modern Applied Statistics with S. Fourth edition. Springer. Wang, Y.Y. (1971). Probabilities of the type I errors of the Welch tests for the Behrens-Fisher problem. Journal of the American Statistical Association, 66, 605-608. Wichert, S., Fokianos, K., and Strimmer, K. (2004). Identifying periodically expressed transcripts in microarray time series data. Bioinformatics, 20:5-20. Zuker, M. & Stiegler,P. (1981) Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res., 9, 133148. Zuker, M. (2003). 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REFERENCES Index aggregation, 95 Anderson-Darling test, 64 annotation, 104 background correction, 94 Binomial test, 58 BLOSUM50, 185 bootstrap, 127, 132 box-and-wiskers-plot, 20 calculator, 4 chi-squared distribution, 37 chi-squared test, 59 classiﬁcation tree, 150 confusion table, 158 construct a sequence, 4 correlation coeﬃcient, 130 data matrix, 6 data vector, 5 density, 41 design matrix, 101 dinucleotide, 176 distance, 118 downloading sequences, 174 F-distribution, 40 F-test, 57 Fisher test, 62 frequency table, 17 genBank, 18 gene ﬁltering, 97 gene ontology, 107 GO, 107 gol.fac, 11 Golub et al. (1999) data, 10 grep, 12 help, 3 histogram, 19 homoscedasticity, 85 install R, 1 installing Bioconductor, 2 installing R, 2 interquartile range, 25 k-means cluster analysis, 125 Kruskal-Wallis test, 87 linear model, 74 matrix computations, 8 mean, 24 median, 24 median absolute deviation, 25 misclassiﬁcation rate, 158 mismatch, 91 model matrix, 101 Needleman-Wunsch, 184 neural network, 162 normal distribution, 35 263 264 INDEX normality of residuals, 85 normality test, 63 normalization, 94 one sample t-test, 51 one sided hypothesis, 48 one-way analysis of variance, 77 packages, 2 perfect match, 91 Phylogenetic tree, 203 predictive power, 147 principal components analysis, 133 Quantile-Quantile plot, 22 quartile, 20 query language, 173 receiver operator curve, 148 rma, 95 running scripts, 13 sample variance, 25 sensitivity, 147 Shapiro-Wilk test, 63 signiﬁcance level, 48 single linkage cluster analysis, 121 speciﬁcity, 147 standard deviation, 25 stripchart, 19 support vector machine, 161 T-distribution, 39 training set, 159 triangle inequality, 118 two sided hypothesis, 48 two-sample t-test, 54 validation set, 159 Wilcoxon rank test, 65 Z-test, 48 ii Preface The purpose of this book is to give an introduction into statistics in order to solve some problems of bioinformatics. Statistics provides procedures to explore and visualize data as well as to test biological hypotheses. The book intends to be introductory in explaining and programming elementary statistical concepts, thereby bridging the gap between high school levels and the specialized statistical literature. After studying this book readers have a sufﬁcient background for Bioconductor Case Studies (Hahne et al., 2008) and Bioinformatics and Computational Biology Solutions Using R and Bioconductor (Genteman et al., 2005). The theory is kept minimal and is always illustrated by several examples with data from research in bioinformatics. Prerequisites to follow the stream of reasoning is limited to basic high-school knowledge about functions. It may, however, help to have some knowledge of gene expressions values (Pevsner, 2003) or statistics (Bain & Engelhardt, 1992; Ewens & Grant, 2005; Rosner, 2000; Samuels & Witmer, 2003), and elementary programming. To support self-study a suﬃcient amount of challenging exercises are given together with an appendix with answers. The programming language R is becoming increasingly important because it is not only very ﬂexible in reading, manipulating, and writing data, but all its outcomes are directly available as objects for further programming. R is a rapidly growing language making basic as well as advanced statistical programming easy. From an educational point of view, R provides the possibility to combine the learning of statistical concepts by mathematics, programming, and visualization. The plots and tables produced by R can A readily be used in typewriting systems such as Emacs, L TEX, or Word. Chapter 1 gives a brief introduction into basic functionalities of R. Chapter 2 starts with univariate data visualization and the most important descriptive statistics. Chapter 3 gives commonly used discrete and continuous distributions to model events and the probability by which these occur. These distributions are applied in Chapter 4 to statistically test hypotheses from bioinformatics. For each test the statistics involved are brieﬂy explained and its application is illustrated by examples. In Chapter 5 linear models are explained and applied to testing for diﬀerences between groups. It gives a basic approach. In Chapter 6 the three phases of analysis of microarray data (preprocessing, analysis, post processing) are brieﬂy introduced and illustrated by many examples bringing ideas together with R scrips and interpretation of results. Chapter 7 starts with an intuitive approach into Euclidian distance iii and explains how it can be used in two well-known types of cluster analysis to ﬁnd groups of genes. It also explains how principal components analysis can be used to explore a large data matrix for the direction of largest variation. Chapter 8 shows how gene expressions can be used to predict the diagnosis of patients. Three such prediction methods are illustrated and compared. Chapter 9 introduces a query language to download sequences eﬃciently and gives various examples of computing important quantities such as alignment scores. Chapter 10 introduces the concept of a probability transition matrix which is applied to the estimation of phylogenetic trees and (Hidden) Markov Models. R commands come after its prompt >, except when commands are part of the ongoing text. Input and output of R will be given in verbatim typewriting style. To save space sometimes not all of the original output from R is printed. The end of an example is indicated by the box . In its Portable Document Format (PDF)1 there are many links to the Index, Table of Contents, Equations, Tables, and Figures. Readers are encouraged to copy and paste scripts from the PDF into the R system in order to study its outcome. Apart from using the book to study application of statistics in bioinformatics, it can also be useful for statistical programming. I would like to thank my colleges Joop Bouman, Sven Warris and Jan Peter Nap for their useful remarks on parts of an earlier draft. Many thanks also go to my students for asking questions that gave hints to improve clarity. Remarks to further improve the text are appreciated. Wim P. Krijnen Hanze University Institute for Life Science and Technology Zernikeplein 11 9747 AS Groningen The Netherlands w.p.krijnen@pl.hanze.nl Groningen October 2009 c This document falls under the GNU Free Document Licence and may be used freely for educational purposes. 1 iv Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Brief Introduction into Using R 1.1 Getting R Started on your PC . . . . 1.2 Getting help . . . . . . . . . . . . . . 1.3 Calculating with R . . . . . . . . . . 1.4 Generating a sequence and a factor . 1.5 Computing on a data vector . . . . . 1.6 Constructing a data matrix . . . . . 1.7 Computing on a data matrix . . . . . 1.8 Application to the Golub (1999) data 1.9 Running scripts . . . . . . . . . . . . 1.10 Overview and concluding remarks . . 1.11 Exercises . . . . . . . . . . . . . . . . iii 1 1 3 4 4 5 6 8 10 12 13 14 17 17 17 18 19 20 22 24 24 25 26 26 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Data Display and Descriptive Statistics 2.1 Univariate data display . . . . . . . . . . 2.1.1 Pie and Frequency table . . . . . 2.1.2 Plotting data . . . . . . . . . . . 2.1.3 Histogram . . . . . . . . . . . . . 2.1.4 Boxplot . . . . . . . . . . . . . . 2.1.5 Quantile-Quantile plot . . . . . . 2.2 Descriptive statistics . . . . . . . . . . . 2.2.1 Measures of central tendency . . 2.2.2 Measures of spread . . . . . . . . 2.3 Overview and concluding remarks . . . . 2.4 Exercises . . . . . . . . . . . . . . . . . . v . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Estimation and Inference 4. .3 Overview and concluding remarks . . . 3. . . . . . . . . . . . . . . 5. . . . . . . . . . . . . . . . . . . . . . . . 4. . . . . .2 Application of tests to a whole set gene expression 4. . .1. . . . . . . . . . . . .1 Deﬁnition of linear models . . . 5 Linear Models 5. . . . .1 Discrete distributions . .2. . . . . . . . . . . . .1. . . . . . . . . . . . . 3.3 T-Distribution . . . .3 Two-way analysis of variance . . . .2. . . . . . . . . . . . . . . . . . . . .1.5 Robust tests . . . . . . . 4. 3. . . . . . . . . . . . . . . 3. . 4.vi 3 Important Distributions 3. . . . . . . . . . . . . . . . . . . . . .2 One-way analysis of variance . . 5. . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Statistical hypothesis testing . . . .4 Checking assumptions . . . 4. . . . . . . . .2 Continuous distributions . . . . . . . . . . . . . . . . . . 4.1. . . . . . . . . . . .1. . . . . . . . . . . . . . . . . . . . . . . . . . .5 Plotting a density function 3. . 3. . 4. . . . . . . 4. .1. . . . . . . . .1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Two-sample t-test with unequal variances 4. . . . . . .2. . . . . . . . . .7 Chi-squared test . . . .2 Chi-squared distribution . . . . . . . .10 Wilcoxon rank test . . . . . . . . . . . . .1. . . . . . . .4 Two sample t-test with equal variances . . . . 3.6 Binomial test . . . . . . . CONTENTS 31 31 31 34 35 37 39 40 41 42 43 47 47 48 51 54 56 57 58 59 63 64 65 66 68 69 73 74 77 83 85 86 88 88 .1 Binomial distribution .1. . . . .5 F-test on equal variances . . . . . . . 4. . . . . . . . . . . . . . . . . . . . . . . . . .4 Exercises . . . . . . . . . . . . . . . . . . . . . . . . . . .4 Exercises . . . . . . . .7 Exercises . . . . . . . . . . . . . .2 One Sample t-Test . . . . . . . . . . . . . . . . . . . .9 Outliers test . . . . . . . . . . . . . . . 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . data . . . . . .1. . . . . . .1 Normal distribution .2. . . . . . . . . . . . . . . . . 5. . . . . . . . . . . . . . . . . . . 4. .1. . .2. . . 5. . . . 3. .3 Overview and concluding remarks 3. . . . . . . . . . .1 The Z-test . . . .6 Overview and concluding remarks 5. . . . . . . .4 F-Distribution .8 Normality tests . 5. . . . . . . . . . . . . . . .2. . 162 . . . . . .3 Classiﬁcation trees . .2 Preprocessing methods . . . . . . .7 Overview and concluding remarks 8. . . 6. . . . . . . . . . . . 7. . . . . . . . . . . . . . . . . . . . . . 167 . . . . . . .5 Overview and concluding remarks 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 . .2 ROC types of curves . . . . . . . . . . . .CONTENTS 6 Micro Array Analysis 6. . . . . . . . . . . . . . . . . 125 . . . . . .5 Neural Networks . . . . . . . . . . . .8 GO parents and children . . . 142 145 . . . . . . . . . .7 Searching GO numbers and evidence 6. . . 6. . . . . . . . . 8. . . . . . . . . . . 6. . .4 Applications of linear models . . . . . . .10 Signiﬁcance per chromosome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 Gene ﬁltering . . 7. . . . . 8. . . . . 141 . . . . . . . . . . 146 . 8. . . . . . . . . . . . . . . . 6. . . . . . . 133 . . . . . . . . . 8. . . . 130 . . .2 Two types of Cluster Analysis . . . . . . 7. . . . . . . . . . . 174 9. 6. . . . . . 164 . . . 6. . . . . . . . . . . . . . . . . 8. . . . . .2 k-means . . . 121 . . . . . . . . . .12 Exercises . . . . . . . . . . . .1 Distance . . . 6. . . . . . 7. . . . . . vii 91 91 94 97 100 104 106 107 108 109 110 112 112 .2 Getting information on downloaded sequences . .1 Single Linkage . . . . . . .11 Overview and concluding remarks . . . . . . . . . . . . . . 117 .3 The correlation coeﬃcient . . . . . . . .6 Using annotation to search literature 6. . . . . . . . . 176 . . 150 . . 160 . . . . . . . . . . . . 7 Cluster Analysis and Trees 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .6 Exercises . . . . . . .5 Searching an annotation package . . . . . . . . . . . . 7. . . . .9 Gene ﬁltering by a biological term . . . . .3 Computations on sequences . . . . . . . . . .4 Principal Components Analysis . . . . . . . .1 Using a query language .1 Probe data . . . 6. . . . . . . . . . . . . 173 9. . 8 Classiﬁcation Methods 8. . . . . . . . .4 Support Vector Machine . . . . 8. . . . . . . . . . . . . . . . . . . . . . 6. . . . . . . 7. . . . . . . . . . . .1 Classiﬁcation of microRNA . . . . . . . . . . . . . . . . . . . . . . . . . . .8 Exercises . . . . . . . . . . . . . . . . . . . . . . 118 . . . . . . . . .2. . .6 Generalized Linear Models . . . . . . . . . . . . . . . 167 9 Analyzing Sequences 173 9. . 147 . . . . . . . . . . . .5 9. . . . . . . . . . . . .3 Properties of the transition matrix 10. . . . . .4 Stationary distribution . . Pairwise alignments . 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Appendix . . . . . .8 Overview and concluding remarks . . . . . . . . . . . . . . .6 Hidden Markov Models . . remarks . . . . . 10. . . . CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . .4 9. . . . . .5 Phylogenetic distance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Probability transition matrix . . . . . . . . . . . . . 181 182 189 189 193 193 194 199 201 203 209 213 214 214 219 257 10 Markov Models 10. . . . . . . . . . . . . . 10. . . . . .viii 9. . . . . . . . . . . 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 Matching patterns . . 10. . . . . . . . 10. . . Overview and concluding Exercises . . . . . . . . .9 Exercises . . . . . . . 10. A Answers to exercises B References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 Random sampling .6 9. 10 . .2 2. . . . . . . . . . . . . . . . . . . . . . . . Histogram of ALL expression values of gene CCND3 Cyclin D3. . . .5. . . . . . . . . . . . . . . . . . . . . . 81 Plot of Ets2 expression values for three patient groups. . . . . . Distribution of F26. . . 50 Acceptance and rejection regions of the T5 -test. 5 χ2 distribution. . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Plot of gene expression values of CCND3 Cyclin D3. . . . . . . . . . . .10 4. . . . 81 ix . . . . . Density of F26. . . . .4 3. Binomial cumulative probabilities with n = 22 and p = 0.2 4. . . . . . . Graph of normal distribution with mean 1. . . . . . .1 3. .7. . . . . . .2 3. .7 . . . . . . . . . . . . .4 2. .6 3. . . . . . . . . . . . .3 5. Binomial probabilities with n = 22 and p = 0. . . . . . . . . . .9 and standard deviation 0. . . . . . . . 20 20 21 21 23 29 . . . .1 5. . . . . . . . . . . . .1 4. . 34 . . 52 Rejection region of χ2 -test. . . . . 36 . . . . . . . 36 38 38 39 39 41 41 Acceptance and rejection regions of the Z-test. . . . . .10 . . . 5 Density of T10 distribution. . . . . Graph of normal density with mean 1. . . . .5 3. . . . . 34 . . . . . . . . . . . . . . . .8 3. . . . . . . . . .List of Figures 2. . . . . . . Stripchart of gene expression values of CCND3 Cyclin D3 for ALL and AML patients. Boxplot of ALL and AML expression values of gene CCND3 Cyclin D3.9 3. . . . . . . . . . . . . . . . .5 2. . . Q-Q plot of ALL gene expression values of CCND3 Cyclin D3.7 3. . . Distribution function of T10 . . . .5.3 2. . . . . . .9 and standard deviation 0. . .1 2. . . . . . . . . . . . . . . . . 59 3 Plot of SKI-like oncogene expressions for three patient groups. . . . . . .3 3. . . Boxplot with arrows and explaining text. . . . . . . .6 3. . χ2 -density. . . . . . . . . . . . . . . 126 Tree of single linkage cluster analysis. . .10 7. . . . . . . . . . . . . . . . and AML patients. . . . . . . . . 122 Tree of single linkage cluster analysis. . . . B. .13 7. . . . . . . . . . . . . 97 . . . . . . . . . . . . . .x 6. and C for the classiﬁcation of ALL1. . .2 7. .1 6. . . . . . . . . 100 . . . . . . . . . . . . . . . . Boxplot of expression values of gene a for each leukemia class. . . . . 122 Example of three without clusters. . . .4 8. . . . . .5 6. . . . .5 8. . .5 7. . .11 7. . . . . . . . . . . . 138 Single linkage cluster diagram of selected gene expression values. . . . . . . . . . . . Classiﬁcation tree of expression values from gene A. .7 7.2 6. .8 7. 100 7. . . . . . . . . . . . . . . Boxplot of the ALL1/AF4 patients. . . .1 8. . . . . . . . . . . .14 8. 130 Vectors of linear combinations. .1 7. . . . . . Boxplot of expression values from gene CCND3 Cyclin D3 for ALL and AML patients . . . . . . . 149 149 151 151 154 154 156 . . . . . . . . . . . . . . . . . .4 7. . . . . . .3 8. . . . Boxplot of the ALL1/AF4 patients after median subtraction and MAD division. . . . . . . . . . . . . . . . .2 8. 93 . . 123 Plot of gene ”CCND3 Cyclin D3” and ”Zyxin” expressions for ALL and AML patients. . . . . . . . . . 93 . . . . . . . . Classiﬁcation tree for gene for three classes of leukemia. . . . . . . . Boxplot of expression values of gene a for each leukemia class. . . . .12 7. . . . . . . . . . . . . . ALL2. Boxplot of the ALL1/AF4 patients after median subtraction and MAD division. .7 Plot of ﬁve points to be clustered. 124 K-means cluster analysis. . . . . . . . 144 ROC plot for expression values of CCND3 Cyclin D3. . . . 126 Plot of kmeans (stars) cluster analysis on CCND3 Cyclin D3 and Zyxin discriminating between ALL (red) and AML (black) patients. . . . . . . . .6 8. . . . . . . 124 Single linkage cluster diagram from gene ”CCND3 Cyclin D3” and ”Zyxin” expressions values. . . . . . . . . ROC plot for expression values of gene Gdf5. 135 Scatter plot of selected genes with row labels on the ﬁrst two principal components. . . . . . . . . . .138 Biplot of selected genes from the golub data. . . . .3 6. . . . . . . .B. . .6 7. . .9 LIST OF FIGURES Mat plot of intensity values for a probe of MLL. . . .4 6. . . . . Density of MLL. . . . . . . . . . . . . . . . . .6 7. . . 123 Three clusters with diﬀerent standard deviations. . . . 97 . .3 7. 135 First principal component with projections of data. . Venn diagram of seleced ALL genes. . . .B data. . . . . . . . . . .10 Variable importance plot on ALL B-cell 123 data. . . . . . . . . 178 Frequency plot of amino acids from accession number AF517525. . .1 9. . . 9. . . . . . . . .1 Graph of probability transition matrix .3 xi . . . . . . . . . . .LIST OF FIGURES 8. . 8.179 10. . . . . . . .CCND3.8 Classiﬁcation tree of expression values from gene CCND3 Cyclin D3 for classiﬁcation of ALL and AML patients. . . . . . . . . .11 Logit ﬁt to the CCND3 Cyclin D3 expression values. .179 Frequency plot of amino acids from accession number AL160163. . . 8. . . . . . . . .2 9.3 Tree according to GTR model. . 8.2 Evaluation of models by AIC . . . . . . . . . . . . . . . . . . 216 10. . . . . . . . . . . . .9 rpart on ALL B-cel 123 data. . . . . . 196 10. 156 159 159 171 G + C fraction of sequence ”AF517525. . . .CCND3” along a window of length 50 nt. 217 . . .CCND3. . . . . . xii LIST OF FIGURES . . . .6. . . 146 Ordered expression values of gene CCND3 Cyclin D3. . . . . . number of true positives. . 170 BLOSUM50 matrix. . . index 2 indicates ALL.3 7. 1 indicates AML. . . .1 8. . . . . . . . . .List of Tables 2. . . . . . . . . . . 18 Discrete density and distribution function values of S3 . . . . . . . 43 Data set for principal components analysis. . . . 33 Built-in-functions for random variables used in this chapter. 186 9. . . . .1 3. . . . number of false positives.1 xiii . with p = 0. . . . . . . . . .01. mean. . 134 Frequencies empirical p-values lower than or equal to 0. . . . .2 A frequency table and its pie of Zyxin gene. . . . . . . . . . . . . . 42 Density. . and variance of distributions used in this chapter. . . .1 8. . . . . . . true positive rate. false positive rate. cutoﬀ points. . . . .2 3. . . . . .1 3. . . . xiv LIST OF TABLES . With respect to gene expressions the data vectors are placed one beneath the other to form a data matrix with the genes as rows and the patients as columns. The standard installation of R makes basic packages available such as base and stats. After a little patience you should be able to start R (Ihaka & Gentleman. 1. A larger example consists of the classical Golub et al.1 Getting R Started on your PC You can downloaded R freely from http://cran. several basic illustrations of this are given. This will solve the practical issues to follow the stream of reasoning. speciﬁc for our purposes. (1999) data.Chapter 1 Brief Introduction into Using R To get started a gentle introduction to the statistical programming language R will be given (R Development Core Team. 1996) after which a screen is opened with the prompt >. The idea of a data matrix is extensively explained and illustrated by several examples. In particular. Click on your favorite operating system (Windows. and how to perform simple calculations.r-project. which will be analyzed frequently to illustrate statistical procedures.org. The input and output of R will be displayed in verbatim typewriting style. All useful functions of R are contained in libraries which are called ”packages”.r-project.org it can be seen that R has a huge number of packages available for a wide scale 1 . how to obtain help. Since many computations are essentially performed on data vectors. Linux or MacOS) and simply follow the instructions. it is brieﬂy explained how to install R and Bioconductor. From the button Packages at cran. 2009). informatics. For further information the reader is referred to www.org.gmane. Bioconductor is primarily based on R and can be installed.html http://dir. http://mccammon. et.biology. you can use the following.R") > biocLite() Then to download the ALL package from a repository to your system.edu/software.1)) In the sequel we shall often use packages from Bioconductor. to produce a nice plot of the outcome of throwing twelve times with a die.r-project.bioconductor.org". + dep=TRUE) This installs the package TeachingDemos developed by Greg Snow from the repository http://cran.edu/~bgrant/bio3d/user_guide/user_guide. > biocLite("ALL") > library(ALL) > data(ALL) These data will be analyzed extensively later-on in Chapter 5 and 6.repo="http://cran. For instance.conductor 1 .ucsd. By setting the option dep to TRUE the packages on which the TeachingDemos depend are also installed. This is strongly recommended! Alternatively. > source("http://www.science. al.html http://rafalab. 2004) available for usage.org/gmane. in the Windows application of R you can simply click on the Packages button at the top of your screen and follow the instructions. you can use the following.org/biocLite.packages(c("TeachingDemos").jhsph.2 CHAPTER 1.r-project.bioconductor. > install. To follow the book it is essential to install Bioconductor on your PC or network.org or to several other URL’s1 . a very useful open source software project for the analysis and comprehension of genomic data. General help on loaded Bioconductor packages becomes available by openVignette(). To download a speciﬁc package you can use the following. as follows. and to make the ALL data (Chiaretti. BRIEF INTRODUCTION INTO USING R of statistical procedures. After installing you have to load the package in order to use its functions. > library(TeachingDemos) > plot(dice(12. to load it. csv. For instance. simply type apropos("if"). The golub data become available by the following. Type objects() or ls() to view the currently loaded objects.2. or simply click on the cross in the upper right corner of your screen.org.r-project. a list probably growing soon to be large.2 Getting help All functionalities of R are well-organized in so-called packages. At http://www.org you can use R site Functions to read data into R are read. because these contain many basic functionalities. Such is given by example(boxplot). it is convenient to have an example showing output (a plot) and programming code.table or read. ”R Installation and Administration”. Further help can be obtained from http://cran. The packages stats and base are automatically installed. Use the function library() to see which packages are currently installed on your operating system.2 > library(multtest) > data(golub) R is object-oriented in the sense that everything consists of objects belonging to certain classes. ?sum gives details on summation. Its ”contributed” page contains well-written freely available on-line books3 and useful reference charts4 . and ”R Data Import/Export”.r-project. 3 ”R for Beginners” by Emmanuel Paradis or the ”The R Guide” by Jason Owen 4 ”R reference card” by Tom Short or by Jonathan Baron 2 . ”The R Language Deﬁnition”. When you are starting with a new concept such as ”boxplot”. To obtain an overview of the content of a package use ls(package:stats) or library(help="stats"). (1999) data. Type help. To prevent conﬂicting deﬁnitions.1. 1. see also Section 1. To quit a session. Type class(golub) to obtain the class of the object golub and str(golub) to obtain its structure or content. GETTING HELP 3 In this and the following chapters we will illustrate many statistical ideas by the Golub et al. The function history can be useful for collecting previously given commands. type q().8. Help on the purpose of speciﬁc functions can be obtained from the (package) manual by typing a question mark in front of a function. In case you are seeking help on a function which uses if. it is wise to remove them all at the end of a session by rm(list=ls()).start() to launch an HTML page linking to several wellwritten R manuals such as: ”An Introduction to R”. see also the ”The R Data Import/Export manual”. There are a number of useful URL’s with information on R. > 2+3 [1] 5 In many calculations the natural base e = 2. Rseek.4 CHAPTER 1. indeed. we need to generate sequences of numbers.4 Generating a sequence and a factor In order to compute so-called quantiles of distributions (see e.edu/~tgirke/Documents/R_BioCond/R_BioCondManual. for any value of x.g. > exp(1) [1] 2.3 Calculating with R R can be used as a simple calculator. we have ex =exp(x).html 6 The argument of functions is always placed between parenthesis ().4) or plots of functions.ucr. The easiest way to construct a sequence of numbers is by > 1:5 [1] 1 2 3 4 5 We mention in particular: http://faculty. or other useful search engines.6 So.1. 5 .718282 of exponential functions is used. to add 2 and 3 we simply insert the following.5 1.718282 To compute e2 = e · e we use exp(2). Section 2. BRIEF INTRODUCTION INTO USING R search. For instance. Such type of functions can be called as follows. The sum 1 + 2 + 3 + 4 + 5 can be computed by > sum(1:5) [1] 15 and the product 5! = 5 · 4 · 3 · 2 · 1 by > prod(1:5) [1] 120 1. 5. > seq(0. called a “factor”.1. Chap. 8) for a full explanation of experiments and statistical principles of design.c(1. and 1. 7 .7 0.0 0. as follows. This can be illustrated by a simple example on expression values of a gene. Each of these levels has ﬁve repeats corresponding to the number of observations (patients) within each level (type of disease). > gene1 <. the corresponding factor can be generated as follows.9 1.00 1.5 Computing on a data vector A data vector is simply a collection of numbers obtained as outcomes from measurements.1.5 0. > factor <. which allows for various sizes of steps to be chosen. We shall further illustrate the idea of a factor soon because it is very useful for purposes of visualization.1.25 from the persons ”Eric”. and ”Anna” are available. in order to compute percentiles of a distribution we may want to generate numbers between zero and one with step size equal to 0.2 0. Samuals & Witmer (2003. for instance. To store these in a vector we use the concatenate command c().5. COMPUTING ON A DATA VECTOR 5 This sequence can also be produced by the function seq.7 When.1.50 1.50.1 0.6 0. 1.4 0.g. ”Peter”.3 0.00.0. for each of three experimental conditions there are measurements from ﬁve patients. For instance.0 For plotting and testing of hypotheses we need to generate yet another type of sequence. It is designed to indicate an experimental condition of a measurement or the group to which a patient belongs.gl(3.25 See e.5) > factor [1] 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 Levels: 1 2 3 The three conditions are often called “levels” of a factor.1) [1] 0.8 0.25) > gene1 [1] 1. Suppose that gene expression values 1.1. 1. x2 = 1.20. the sum of the weights can be expressed as n xi = 3. To compute the sum.1.55. mean.entry you can open and edit a screen with the values of a matrix.-1. and x3 = 1.50. To illustrate this by a small example suppose that we have the following expression values on three genes from Eric.-1. > sum(gene1) [1] 3.6 Constructing a data matrix In various types of spreadsheets it is custom to store data values in the form of a matrix consisting of rows and columns.25) > gene4 <.25 > sqrt(sum((gene1-mean(gene1))^2)/2) [1] 0.00) > gene3 <. 1.c(-1.75 > mean(gene1) [1] 1.25 By deﬁning x1 = 1.-1. The mathematical summation symbol is i=1 in R language simply sum.75. and Anna. and standard deviation of the gene expression values we use the corresponding built-in-functions.6 CHAPTER 1.25 i=1 and the sample standard deviation as 3 s= i=1 (xi − x)2 /(3 − 1) = 0. 8 . Peter.00) By the function data.25.00.c(-1.25 > sum(gene1)/3 [1] 1.30. BRIEF INTRODUCTION INTO USING R Now we have created the object gene1 containing three gene expression values.25 > sd(gene1) [1] 0.c(1.25.50.8 > gene2 <. The mean is denoted by x = 3 xi /3 = 1.-1.35.1. In bioinformatics gene expression values (from several groups of patients) are stored as rows such that each row contains the expressions values of the patients corresponding to a particular gene and each column contains all gene expression values for a particular person.10. then type gendat[1."gene4").2].50 1.]. Consider the following script. + c("Eric".30 -1.2]. To see the content of the just created object gendat. The names of the rows and columns are attached by the dimnames parameter. . we print it to the screen. use gendat[.30 -1.00 1.1."Peter". > write.55 1.list(c("gene1"."gene2". as follows.25 9 Indices referring to rows.25 1.gene4).Rdata") > gendatread Eric Peter Anna gene1 1.20 -1. To do so we construct the following list. > gendat gene1 gene2 gene3 gene4 Eric Peter Anna 1. For the second column. Thus.read.file="D:/data/gendat. CONSTRUCTING A DATA MATRIX 7 Before constructing the matrix it is convenient to add the names of the rows and the columns. > rowcolnames <. The gene vectors are placed in the matrix as rows.table(gendat.table("D:/data/gendat.Rdata") > gendatread <."gene3". + byrow=TRUE. columns. dimnames = rowcolnames) Here. or elements are always between square brackets []. ncol=3.25 -1.matrix(c(gene1.50 -1. nrow indicates the number of rows and ncol the number of columns. if you want to print the second element of the ﬁrst row to the screen. > gendat <. the ﬁrst of which refers to rows and the second to columns9 .gene2.50 1. It may be desirable to write the data to a ﬁle for using these in a later stage or to send these to a college of yours.00 1. If you want to print the ﬁrst row.00 A matrix such as gendat has two indices [i.j].10 -1. Now we can construct a matrix containing the expression values from our four genes."Anna")) After the last comma in the ﬁrst line we give a carriage return for R to come up with a new line starting with + in order to complete a command.6. then use gendat[1. nrow=4.gene3.35 1. Such type of computations on a data matrix can be accomplished by “for loops”. > apply(gendat.166667 It frequently happens that we want to re-order the rows of a matrix according to a certain criterion.0125 0. To do so we specify the name of the matrix. the values in a certain column vector.25 gene4 -1.csv.50 -1. 10 . the mean of each gene (row) can be computed. it is convenient to store these in a vector and to use the function order. However.00 An alternative is to use write.mean) gene1 gene2 gene3 gene4 1.30 -1.8 CHAPTER 1. and the name of the function. or search the internet by the key ”r wiki matrix”.10 1.1.2.7 Computing on a data matrix Means or standard deviations of rows or columns are often important for drawing biologically relevant conclusions.0625 0.order(meanexprsval.mean) Eric Peter Anna 0.55 1. more speciﬁcally. Chapter 3 of the book ”R for Beginners”.mean) > o <. To illustrate this we compute the mean of each person (column). BRIEF INTRODUCTION INTO USING R gene2 1.20 -1.0750 Similarly.decreasing=TRUE) > o [1] 2 1 4 3 For more see the ”R Data import/Export” manual. or.30 gene3 -1.1.apply(gendat.283333 -1.250000 1. to re-order the matrix gendat according to the row means. > meanexprsval <. indicate rows or columns (1 for rows and 2 for columns). it is much more convenient to use the apply functionality on a matrix.10 -1. For instance. > apply(gendat.35 1.400000 -1. 30 A second way is to use the row names as an index. we can re-order the whole matrix by specifying o as the row index.00 1. followed by gene4 with -1.25 gene4 -1. we may evaluate whether the row mean is positive. then gene1 with 1.30 -1. > gendat[c("gene1".25 gene2 1. 11 You can also use functions like sort or rank. > gendat[c(1.00 1.16 and."gene2"). Now that we have collected the order numbers in the vector o.30 A third and more advanced way is to use an evaluation in terms of TRUE or FALSE of logical elements of a vector. For instance.7.55 1. .25 Another frequently occurring problem is that of selecting genes with a certain property.2).25 gene2 1.] Eric Peter Anna gene1 1. ﬁnally.20 -1.25. We illustrate this by several methods to select genes with positive mean expression values.50 -1.55 1.2) as a row index.1.28.11 > gendat[o.10 -1.50 1.] Eric Peter Anna gene2 1.00 1.35 1.30 gene1 1.55 1.4. COMPUTING ON A DATA MATRIX 9 Thus gene2 appears ﬁrst because it has the largest mean 1.35 1.50 1. A ﬁrst method starts with the observation that the ﬁrst two rows have positive means and to use c(1.00 gene3 -1.] Eric Peter Anna gene1 1.50 1. > meanexprsval > 0 gene1 gene2 gene3 gene4 TRUE TRUE FALSE FALSE Now we can use the evaluation of meanexprsval > 0 in terms of the values TRUE or FALSE as a row index.35 1. gene3 with -1. To load the data and to obtain relevant information from row 1042 of golub.00 1. > library(multtest).gnames of which the columns correspond to the gene index. which is part of Bioconductor.10 CHAPTER 1. A selection of the set is called golub and is contained in the multtest package. > nrow(golub) [1] 3051 > ncol(golub) [1] 38 12 The data are pre-processed by procedures described in Dudoit et al. The gene names are collected in the matrix golub.] [1] "2354" "CCND3 Cyclin D3" "M92287_at" The data are stored in a matrix called golub. row name or value on a logical variable. The expression values of this gene are collected in row 1042 of golub. BRIEF INTRODUCTION INTO USING R > gendat[meanexprsval > 0. This illustrates that genes can be selected by their row index. . (1999) are among the classical in bioinformatics. respectively.8 Application to the Golub (1999) data The gene expression data collected by Golub et al.cl. respectively. and Name. where ALL is indicated by 0 and AML by 1. ID.] Eric Peter Anna gene1 1.gnames.35 1.25 gene2 1. The number of rows and columns can be obtained by the functions nrow and ncol. We shall ﬁrst concentrate on expression values of a gene with manufacturer name "M92287_at". which is known in biology as "CCND3 Cyclin D3".50 1.55 1.30 Observe that this selects genes for which the evaluation equals TRUE. 1. The tumor class is given by the numeric vector golub.gnames[1042. data(golub) > golub. The data consist of gene expression values of 3051 genes (rows) from 38 leukemia patients12 . Twenty seven patients are diagnosed as acute lymphoblastic leukemia (ALL) and eleven as acute myeloid leukemia (AML). use the following. (2002). we have to refer to the ﬁrst twenty seven elements of row 1042. APPLICATION TO THE GOLUB (1999) DATA 11 So the matrix has 3051 rows and 38 columns.63637 To print the expression values of gene CCND3 Cyclin D3 to the screen only for the ALL patients.82667 1.12758 -0. labels = c("ALL".fac and is constructed from the vector golub.83051 2. The values of the ﬁrst column can be printed to the screen by the following. > golub[. > gol.52405 So 1. as follows.fac=="ALL" returns TRUE for the ﬁrst twenty seven values and FALSE for the remaining eleven.88941 0. We may now print the expression values of gene CCND3 Cyclin D3 (row 1042) to the screen.10892 [9] 2.96403 1. This is useful as a column index for selecting the expression values of the ALL patients.81649 1.59385 2.32551 2.17622 [17] 1.52405 is the expression value of gene CCND3 Cyclin D3 from patient number 2.2] [1] 1.1:27] However. the labels correspond to the two tumor classes.cl.85111 2. A possibility to do so is by the following.10546 [25] 1.factor(golub. see also dim(golub). Recall that the ﬁrst index refers to rows and the second to columns. > golub[1042.80861 2.31428 0. as follows. The expression values of gene CCND3 Cyclin D3 from the ALL patients can now be printed to the screen.74333 2.44562 1.33597 1. The factor will be called gol.8.90496 1.1.52405 1.fac <.12058 2.1] To save space the output is not shown. levels=0:1.78352 0.99391 1. The evaluation of gol. Obviously.02250 1.92776 1. for the work ahead it is much more convenient to construct a factor indicating the tumor class of the patients. This will turn out useful e.] [1] 2.g. Each data element has a row and a column index.37351 1.36844 [33] 1. .42904 1. for separating the tumor groups in various visualization procedures.18119 0.49470 1. Hence.cl.76610 0. > golub[1042.06597 2.27645 1.45827 1. the second value from row 1042 can be printed to the screen as follows.45014 0."AML")) In the sequel this factor will be used frequently.73784 1.83485 0.99927 0. > golub[1042. the expression values of antigen CD33 are available at golub[808.levels=0:1.] and further information on it by golub. several functions of R are inspired by the Linux operating system.gol. The 3051 means are assigned to the vector meanALL. .2]) [1] 808 Hence.fac=="ALL"]. > > > > > > library(multtest). After reading the classical article by Golub et al.gol. we may use the following. Another possibility is to execute a script from a ﬁle. labels= c("ALL". For instance.apply(golub[. 1.cl. or WinEdt for the formulation of several consecutive R commands as separated lines (scripts).fac=="AML"].9 Running scripts It is very convenient to use a plain text writer like Notepad. 1.gol. decreasing=TRUE) print(golub. mean) maml <. BRIEF INTRODUCTION INTO USING R > golub[1042.2]) 13 Indeed.apply(golub[. This can obtained by the grep function.order(abs(mall-maml). Such command lines can be executed by simply using copy and paste into the command line editor of R.13 > grep("CD33". which is strongly recommended. to compute the mean gene expression over the ALL patients for each of the genes.factor(golub.fac=="ALL"] selects the matrix with gene expressions corresponding to the ALL patients. To illustrate the latter consider the following. gene CD33 plays an important role in distinguishing lymphoid from myeloid lineage cells. 1.fac <.gnames[o[1:5]. To perform computations on the expressions of this gene we need to know its row index."AML")) mall <. mean) The speciﬁcation golub[.apply(golub[.]. mean) o <.gnames[. (1999). Emacs. 1.golub.gol. data(golub) gol.fac=="ALL"].gnames[808. Kate.12 CHAPTER 1.fac=="ALL"] For many types of computations it is very useful to combine a factor with the apply functionality. For instance. one becomes easily interested in the properties of certain genes.gol. > meanALL <. assuming some background on biomedical statistics. (on-line) books and R Wiki at hand you have various sources of information to help you along with practical issues. OVERVIEW AND CONCLUDING REMARKS [1] [2] [3] [4] [5] 13 "CST3 Cystatin C (amyloid angiopathy and cerebral hemorrhage)" "INTERLEUKIN-8 PRECURSOR" "Interleukin 8 (IL8) gene" "DF D component of complement (adipsin)" "MPO Myeloperoxidase" The row means of the expression values per patient group are computed and stored in the object mall and maml. Chambers. 2006). Once the script is available for a typewriter it is easy to adapt it and to re-run it. 2000. & Wilks. the name meandif.g. The absolute values of the diﬀerences in means are computed and their order numbers (from large to small) are stored in the vector o.10 Overview and concluding remarks It is easy to install R and Bioconductor. Other treatments go much deeper into programming aspects (Becker. For the sake of illustration we shall work frequently with the data kindly provided by Golub et al. With the reference charts.1.10. (2004). Readers are strongly recommended to trial-and-error with respect to writing programming scripts. & Ripley. R manuals.R").R in the directory D:\\Rscripts\\meandif. The above introduction is of course very brief. R has many convenient built-infunctions for statistical programming. 2002. 1. Although there recently became several GUI’s available. There are book length treatments combining R with statistics (Venables. respectively. To run these it is very convenient to have your favorite word processor available and to use. it can be executed by using source("D:\\Rscripts\\meandif. 1988. Help and illustrations on many topics are available from various sources. for instance. Everitt & Hothorn. Gentleman.R. The corre- . the copy-and-paste functionality. Next. 2008). we shall concentrate on the command line editor because its range of possibilities is much larger. is given by Dalgaard (2002). the names of the ﬁve genes with the largest diﬀerences in mean are printed to the screen. Venables & Ripley. A more extensive introduction into R. (1999) and Chiaretti et al. After saving the script under e. apply. (a) Use apply to compute the standard deviation per gene.fac. gendat Consider the data in the matrix gendat.6. sd. (b) Order the data matrix according to the gene means. Computations on gene standard deviations of the Golub data. ALL. constructed in Section 1. . str. (d) Which gene has the largest standard deviation? 3. (a) Use the function class to ﬁnd the class to which the following objects belong: golub. 1. gol. plot. (b) What is the meaning of the following abbreviations: rm. BRIEF INTRODUCTION INTO USING R sponding scientiﬁc articles are freely available from the web. history.golub.11 Exercises 1. Its small size has the advantage that you can check your computations even by a pocket calculator. (c) For what purpose are the following functions useful: grep. seq. (c) Give the names of the three genes with the largest mean expression value.gnames.14 CHAPTER 1. (b) Use apply to compute the standard deviation of the genes. golub. nrow. (a) Use apply to compute the mean gene expression value. gl. apply. library. Some questions to orientate yourself. Computations on gene means of the Golub data. golub[1. 2. 14 (a) Use apply to compute the standard deviation of the persons. source. sum. 14 Obtaining some routine with the apply functionality is quite helpful for what follows.cl.1]. setwd. (d) Give the biological names of these genes. prod. 4. Having these available may further motivate readers for the computations ahead. (c) Order the matrix according to the gene standard deviations. exp. 11. Load the ALL data from the ALL library and use str and openVignette() for a further orientation.1. (c) How many genes have this property? 5. EXERCISES 15 (b) Select the expression values of the genes with standard deviation larger than two. (c) Three conditions each with ﬁve measurements. Constructing a factor. Construct factors that correspond to the following setting. (b) Five conditions each with three measurements. (c) Do the same for the AML patients. . (b) Find the biological names of the three oncogenes with the largest mean expression value for the ALL patients. (d) Write the gene probe ID and the gene names of the ten genes with largest mean gene expression value to a csv ﬁle. 6. (a) Use exprs(ALL[. Gene means for B1 patients.ALLBT=="B1"] to extract the gene expressions from the patients in disease stage B1. 7. (b) Give the gene identiﬁers of the three genes with the largest mean. (a) An experiment with two conditions each with four measurements. Oncogenes in Golub data. (a) How many oncogenes are there in the dataset? Hint: Use grep. Compute the mean gene expressions over these patients.

16 CHAPTER 1. BRIEF INTRODUCTION INTO USING R .

inter quartile range. and median absolute deviation). A frequency table simply gives the number of occurrences within a category. 2. It quickly answers questions like: How are my data distributed? How can the frequencies of nucleotides from a gene be visualized? Are there outliers in my data? Does the distribution of my data resemble that of a bell-shaped curve? Are there diﬀerences between gene expression values taken from two groups of patients? The most important central tendencies (mean. T }.1. Example 1.Chapter 2 Data Display and Descriptive Statistics A few essential methods are given to display and visualize data. variance. A gene consists of a sequence of nucleotides {A. C. median) are deﬁned and illustrated together with the most important measures of spread (standard deviation.1 Frequency table Discrete data occur when the values naturally fall into categories. 2. These are frequently used by practitioners as well as by experts. The number of each nucleotide can be displayed in a frequency table. This 17 . G.1 Univariate data display To observe the distribution of data various visualization methods are made available.

g..1")))) From the resulting frequencies in Table 2. small boxes.1"). a c A C 410 789 G 573 T 394 t g 2. by which the values of the data are represented as e. DATA DISPLAY AND DESCRIPTIVE STATISTICS will be illustrated by the Zyxin gene which plays an important role in cell adhesion (Golub et al.1: A frequency table and its pie of Zyxin gene.character=TRUE)) pie(table(read.GenBank(c("X94991. Often.1 it seems that the nucleotides are not equally likely. .1” of the species homo sapiens from GenBank. to construct a pie from a frequency table of the four nucleotides.GenBank(c("X94991. 1999). The code below illustrates how to read the sequence ”X94991. Table 2. install.repo="http://cran.org".2 Plotting data An elementary method to visualize data is by using a so-called stripchart. .packages(c("ape").as.r-project.dep=TRUE) library(ape) table(read. A nice way to visualize a frequency table is by plotting a pie. The accession number (X94991.1.1) of one of its variants can be found in a data base like NCBI (UniGene).18 CHAPTER 2.

1. Observe from the latter that one value is small and the other are more or less symmetrically distributed around the mean. 2. To produce two adjacent stripcharts one for the ALL and one for the AML patients. To plot the data values one can simply use plot(golub[1042.levels=0:1.fac=="ALL"]) The function hist divides the data into 5 intervals having width equal to 0. method="jitter") From the resulting Figure 2. UNIVARIATE DATA DISPLAY 19 it is useful in combination with a factor that distinguishes members from diﬀerent experimental conditions or patients groups. indeed. gol.cl. we use the factor called gol. Example 1.fac from the previous chapter. In the resulting plot in Figure 2. Many visualization methods will be illustrated by the Golub et al. (1999) data.fac. see Figure 2.1. labels= c("ALL". We shall concentrate on the expression values of gene "CCND3 Cyclin D3".]). package = "multtest") gol. . Such a plot is called a histogram.2 it can be observed that the CCND3 Cyclin D3 expression values of the ALL patients tend to have larger expression values than those of the AML patients.3 Histogram Another method to visualize data is by dividing the range of data values into a number of intervals and to plot the frequency per interval as a bar. data(golub. It can be observed that the values for patient 28 to 38 are somewhat lower.] ~ gol.3. > hist(golub[1042. Example 1. which are collected in row 1042 of the data matrix golub.factor(golub.fac <."AML")) stripchart(golub[1042. the picture is not very clear because the groups are not plotted separately.1 the vertical axis gives the size of the expression values and the horizontal axis the index of the patients.5.2. A histogram of the expression values of gene "CCND3 Cyclin D3" of the acute lymphoblastic leukemia patients can be produced as follows. but.

25% of the data values lay on the left side of the number x0.1. Similarly.0 1.5 2.2: Stripchart of gene expression values of CCND3 Cyclin D3 for ALL and AML patients.0 1.25 . Figure 2. A vector with gene expression values can be put into increasing order by the function sort. ] 1.5 0 10 20 Index 30 −0.0 0. the third quartile or 75th percentile is the value x0. etc.0 −0. Such a data display is known as a box-and-whisker plot. A popular method to display data is by drawing a box around the ﬁrst and the third quartile (a bold line segment for the median).5 1. and the smaller line segments (whiskers) for the smallest and the largest data values. That is.5 0.0 2. reason for which it is called the ﬁrst quartile or the 25th percentile.5 0. · · · . DATA DISPLAY AND DESCRIPTIVE STATISTICS 2. where x1 is the smallest. The second quartile is the value x0.75 such that 75% of the data is smaller. xn is smaller.5 golub[1042.5 ALL AML Figure 2.0 0.25 be a number for which it holds that 25% of the data values x1 .1: Plot of gene expression values of CCND3 Cyclin D3. 2.5 2. We shall illustrate this by the ALL . x2 is the ﬁrst-to-the smallest. Let x0.50 such that 50% of the data values are smaller.4 Boxplot It is always possible to sort n data values to have increasing order x1 ≤ x2 ≤ · · · ≤ xn .20 CHAPTER 2. Example 1.

5 1. since the two sub-boxes around the median are more or less equally wide.2.0 1. gol.5 ALL AML golub[1042.3: Histogram of ALL expression values of gene CCND3 Cyclin D3. Furthermore.0 0.326 1.4 it can be observed that the gene expression values for ALL are larger than those for AML.5 0. A view on the distribution of the expression values of the ALL and the AML patients on gene CCND3 Cyclin D3 can be obtained by constructing two separate boxplots adjacent to one another.0 −0.5 3.368 21 The second command prints the ﬁrst ﬁve values of the sorted data values to the screen. Figure 2.fac=="ALL"]. x2 = 1.4: Boxplot of ALL and AML expression values of gene CCND3 Cyclin D3. > boxplot(golub[1042.105 1.5 2.sort(golub[1042. decreasing = FALSE) > x[1:5] [1] 0. the data are quite symmetrically distributed around the median.276 1.fac == "ALL"] Figure 2. Example 2. > x <.1. Note that the mathematical notation xi corresponds exactly to the R notation x[i] Histogram of golub[1042.5 1.0 0. gol. .] ~ gol.0 6 1.105.0 2.458.458 1.0 2. so that we have x1 = 0. UNIVARIATE DATA DISPLAY expression values of gene "CCND3 Cyclin D3" in row 1042 of golub. gol. To produce such a plot the factor gol.fac) From the position of the boxes in Figure 2.fac == "ALL"] 12 10 8 Frequency 4 2 0 0.fac is again very useful.5 2. etc.

00 = 0.179.0. Outliers are data values laying far apart from the pattern set by the majority of the data values. and the third x0.75 + 1.75 − x0. A data point x is deﬁned as an outlier point if x < x0. it can be evaluated to what degree the data are normally distributed.928.45827 and 1.0.5 · (x0.25 ).25 = 1.00 = 2. gol.22 CHAPTER 2.458 1.76610.25. These are the smaller values 0. DATA DISPLAY AND DESCRIPTIVE STATISTICS To compute exact values for the quartiles we need a sequence running from 0. The AML expression values have one outlier with value -0.4 it can be observed that there are outliers among the gene expression values of ALL patients.796.fac=="ALL"]).1.fac=="ALL"]).5 is raised to 3. To deﬁne extreme outliers. 2.5 · (x0. The latter can also be obtained by the function min(golub[1042.77.25 − 1.fac=="ALL"]) and max(golub[1042.50 = 1.75 − x0.74333. The implementation in R of the (modiﬁed) boxplot draws such outlier points separately as small circles.5 Quantile-Quantile plot A method to visualize the distribution of gene expression values is by the so-called quantile-quantile (Q-Q) plot.pvec) 0% 25% 50% 75% 100% 0. From Figure 2.766 The ﬁrst quartile x0. In such a plot the quantiles of the gene expression values are displayed against the corresponding quantiles of the normal (bell-shaped).458 and the largest x1. and the largest value 2.25) > quantile(golub[1042. A straight line is added representing points which correspond exactly to the quantiles of the normal distribution. By observing the extent in which the points appear on the line. Note that this is a descriptive way of deﬁning outliers instead of statistically testing for the existence of an outlier.00 to 1. > pvec <. the second x0. or more brieﬂy by range(golub[1042.00 with steps equal to 0. The smallest observed expression value equals x0.seq(0.179 2.25 ) or x > x0.fac=="ALL"].1.75 = 2. gol. the factor 1. To construct such a sequence the function seq is useful.928 2. gol.796 1. That is. gol. the closer the gene .10546.

1. The above example illustrates a case where the degree of non-normality is moderate so that a clear conclusion cannot be drawn. By making the .fac=="ALL"]) From the resulting Figure 2. while a few others are further away. qqnorm(golub[1042. To produce a Q-Q plot of the ALL gene expression values of CCND3 Cyclin D3 one may use the following. gol. Example 1. gol.5: Q-Q plot of ALL gene expression values of CCND3 Cyclin D3.5 −2 1.0 2.fac=="ALL"]) qqline(golub[1042.5 2.2. the more likely it is that the data are normally distributed.0 1. Normal Q−Q Plot Sample Quantiles 0.5 −1 0 Theoretical Quantiles 1 2 Figure 2. UNIVARIATE DATA DISPLAY 23 expression values appear to the line.5 it can be observed that most of the data points are on or near the straight line.

xn is deﬁned as n 1 1 x= xi = (x1 + · · · + xn ). 2. · · · . interquartile range. 2. the central tendency can be described by the mean or the median. Since extreme data values do not inﬂuence the size of the median. the mean is not robust against outliers.fac=="ALL"]) [1] 1. then the mean and the median are equal.50 .89 > median(golub[1042. When the data are symmetrically distributed around the mean. the reader will gather more experience with the degree in which gene expression values are normally distributed. Since it is the sum of all data values divided by the sample size. it is very robust against outliers. Robustness is important in bioinformatics because data are frequently contaminated by extreme or otherwise inﬂuential data values. gol. gol. and the spread by the variance. These will be deﬁned and illustrated.2. n i=1 n Thus the sample mean is simply the average of the n data values. or median absolute deviation. standard deviation. DATA DISPLAY AND DESCRIPTIVE STATISTICS exercises below. To compute the mean and median of the ALL expression values of gene CCND3 Cyclin D3 consider the following.1 Measures of central tendency The most important descriptive statistics for central tendency are the mean and the median.93 . Example 1. The sample mean of the data values x1 . a few extreme data values may largely inﬂuence its size. > mean(golub[1042. In other words.fac=="ALL"]) [1] 1.2 Descriptive statistics There exist various ways to describe the central tendency as well as the spread of data.24 CHAPTER 2. In particular. The median is deﬁned as the second quartile or the 50th percentile. and is denoted by x0.

which is deﬁned as 1 s = n−1 2 n (xi − x)2 = i=1 1 (x1 − x)2 + · · · + (xn − x)2 .2. 2.75 − x0. · · · . DESCRIPTIVE STATISTICS 25 Note that the mean and the median do not diﬀer much so that the distribution seems quite symmetric. 63). The sample standard deviation s is the square root of the sample variance and may be interpreted as the distance of the data values to the mean. the value IQR(x)/1. The median absolute deviation (MAD) is deﬁned as a constant times the median of the absolute deviations of the data from the median (e.2.2. Jureˇkov´ & Picek. gol.368 . The interquartile range is deﬁned as the diﬀerence between the third and the ﬁrst quartile. these are robust against outliers. and the median absolute deviation. > sd(golub[1042. It equals the standard deviation in case the data come from a bell-shaped (normal) distribution (see Section 3. It can be computed by the function IQR(x).fac=="ALL"]) / 1.2. Example 1.349 is a robust estimator of the standard deviation.491 > IQR(golub[1042. These measures of spread for the ALL expression values of gene CCND3 Cyclin D3 can be computed as follows. More speciﬁcally.50 | multiplied by the constant 1. In R it is computed by the c a function mad deﬁned as the median of the sequence |x1 −x0.349 [1] 0. The standard deviation is the square root of the sample variance.4826.50 |. 2006. n−1 Hence.fac=="ALL"]) [1] 0. |xn −x0. gol.284 > mad(golub[1042. Because the interquartile range and the median absolute deviation are based on quantiles. it is the average of the squared diﬀerences between the data values and the sample mean. The variance and the standard deviation are not robust against outliers. the interquartile range. gol.1). p.fac=="ALL"]) [1] 0.g. that is x0.2 Measures of spread The most important measures of spread are the standard deviation.25 .

4 Exercises Since the majority of the exercises are based on the Golub et al. In particular. 2. 2. Are there outliers? .5. If the data are distributed according to a bell-shaped curve.26 CHAPTER 2. Comparing normality for two genes. (c) Comment on the diﬀerences. (1999) data. Venables and Ripley (2002). (a) Compute the mean and the standard deviation for 1. These plots give a useful ﬁrst impression of the degree of (non)normality of gene expression values. 2.5. Figure 2. (b) Compute the mean and the standard deviation for 1. Optimal choices for this are discussed by e. it is easy to produce a pie. 3. 1. 2. 2. 2. the absolute diﬀerences with respect to the median are somewhat smaller. The number of bars can be chosen by the breaks option of the function hist. 30. Illustration of mean and standard deviation.3 Overview and concluding remarks Data can be stored as a vector or a data matrix on which various useful functions are deﬁned.5.g. To stimulate self-study the answers are given at the end of the book. (1999) data. 2.5. To construct the histogram used the default method to compute the number of bars or breaks. then this is often a good strategy. than the root of the squared diﬀerences. histogram. 1. boxplot. (a) Produce a boxplot for the expression values of the ALL patients and comment on the diﬀerences. it is essential to make these available and to learn to work with it. DATA DISPLAY AND DESCRIPTIVE STATISTICS Due to the three outliers (cf. Consider the gene expression values in row 790 and 66 of the Golub et al. or Q-Q plot of a vector of data.4) the standard deviation is larger than the interquartile range and the mean absolute deviation. 1. That is.

Hint: Use a factor for appropriate separation. (b) Rotate the plot to a vertical position and keep it that way for the questions to come. Use the gene expressions "CCND3 Cyclin D3" of Golub et al. (c) Compute the mean and the median for the expression values of the ALL patients and compare these. (1999) data. Eﬀect size. After using the function plot you produce an object on which you can program.6. It measures the mean relative to the standard deviation. An important statistic to measure the eﬀect size which is deﬁned for a sample as x/s. (c) Color the ALL expressions red and AML blue.4. Hint: Use the pch parameter. EXERCISES 27 (b) Produce a QQ-plot and formulate a hypothesis about the normality of the genes. Hint: Use title. (a) Determine the ﬁve genes with the largest eﬀect size of the ALL patients from the Golub et al. 3. 5. (d) Add a title to the plot. . (1999) collected in row 1042 of the object golub from the multtest library. (a) Construct the boxplot in Figure 2.2. Use the gene expressions from "CCND3 Cyclin D3" of Golub et al. (1999) from row 1042 of the object golub of the multtest library. Hint: Use the col parameter. Box-and-Whiskers plot of "CCND3 Cyclin D3". (b) Invent a robust variant of the eﬀect size and use it to answer the previous question. Do this for both genes. Comment on their size. (a) Produce a so-called stripchart for the gene expressions separately for the ALL as well as for the AML patients. (e) Change the boxes into stars. 4. so that is value is large when the mean is large and the standard deviation small. Hint: Store the ﬁnal script you like the most in your typewriter in order to be able to use it eﬃciently later on. Plotting gene expressions "CCND3 Cyclin D3".

(a) Compute the mean and median for gene expression values of the ALL patients.frame(golub)) to produce a box-and-wiskers plot for each column (person). Make a screen shot to save it in a word processor. (b) Do the same for the AML patients and use par(mfrow=c(2. (b) Compute the SD. Hint Hint Hint Hint 1: 2: 3: 4: Use Use Use Use locator() to ﬁnd coordinates of the position of the plot. Are there genes with clear diﬀerences between the groups? 8. report their range and comment on it. (c) Do the same for the wiskers. Descriptive statistics for the ALL gene expression values of the Golub et al.1)) to combine the two plots such that the second is beneath the ﬁrst. Are there outliers? (b) Compute the mean and medians of the persons. xlim to make the plot somewhat wider. text to add information at a certain position. (d) Export your plot to eps format. (1999) data. 7. DATA DISPLAY AND DESCRIPTIVE STATISTICS (b) Add text to the plot to explain the meaning of the upper and lower part of the box. 6. Box-and-wiskers plot of persons of Golub et al. IQR. . Describe what you see. Oncogenes of Golub et al. the IQR and MAD of the persons. Comment of what you observe. (a) Select the oncogens by the grep facility and produce a box-andwiskers plot of the gene expressions of the ALL patients. What do you observe? (c) Compute the range (minimal and maximum value) of the standard deviations. report their range and comment on it. (1999) data. Are the medians of similar size? Is the inter quartile range more or less equal. (a) Use boxplot(data. arrows to add an arrow. (1999) data.28 CHAPTER 2. and MAD for gene expression values of the ALL patients.

4.5 1.0 2.2.6: Boxplot with arrows and explaining text. 0.5 Median 1. EXERCISES 29 2.0 Outlier .5 Figure 2.

DATA DISPLAY AND DESCRIPTIVE STATISTICS .30 CHAPTER 2.

F. T. 2003). healthy-disease. When there are n trials. and the standard deviation σ (sigma).Chapter 3 Important Distributions Questions that concern us in this chapter are: What is the probability to ﬁnd fourteen purines in a microRNA of length twenty two? If expressions from ALL patients of gene CCND3 Cyclin D3 are normally distributed with mean 1.g. then the number of ways to obtain k successes 31 . Only when deemed relevant. 3.5. These distributions have a wealth of applications to statistically testing biological hypotheses. and illustrated.90 and standard deviation 0. purine-pyrimidine. and chi-squared will be elaborated. the density function.1 Discrete distributions The binomial distribution is fundamental and has many applications in medicine and bioinformatics. the distribution function. explained.4? To answer such type of questions we need to know more about statistical distributions (e. are explicitly deﬁned.1 Binomial distribution The binomial distribution ﬁts to repeated trials each with a dichotomous outcome such as succes-failure. the discrete distribution binomial and the continuous distributions normal. what is the probability to observe expression values larger than 2. heads-tails. Samuels & Witmer. In this chapter several important distributions will be deﬁned. In particular. etc. the mean µ (mu).1. 3.

p). np(1 − p) the variance. It is more eﬃcient to compute this by the built-in-density-function dbinom(k. load the TeachingDemos package and use the command vis.140625 = 0. 1 . and p = 0. The binomial probability of k successes out of n consists of the product of this coeﬃcient with the probability of k successes and the probability of n − k failures.5.1)* 0.25^1* 0. taking p for instance near 0. 2003). IMPORTANT DISTRIBUTIONS out of n is given by the binomial coeﬃcient n! .1) and obtain P (X = 1) = 3! 0.1) The collection of these probabilities is called the probability density function. If two carriers of the gen for albinism marry.1) computes the binomial coeﬃcient. k = 1.421875.32 CHAPTER 3. What is the probability for one child out of three to be albino? To answer this question we take n = 3. Then the probability P of the event (X = k) that k successes occur out of n trails can be expressed as P (X = k) = n! pk (1 − p)n−k .75^2 where choose(3. and np(1 − p) the standard deviation.binom().n. For a binomially distributed variable np is the mean. To visualize the Binomial distribution. Let p be the probability of succes in a single trial and X the (random) variable denoting the number of successes. Click on ”Show Normal Approximation” and observe that the approximation improves as n increases. (3.251 0.25 into Equation (3.752 = 3 · 0. for instance to print the values of the probabilities. · · · . k!(n − k)! where n! = n · (n − 1) · · · 1 and 0! = 1 (Samuels & Witmer. Example 2. 1!(3 − 1)! An elementary manner to compute this in R is by > choose(3. n. then each of the children has probability of 1/4 of being albino. 1 Example 1. k!(n − k)! for k = 0.

38 = dbinom(14. with p = 0. The probability of this event can be computed by P (X = 14) = 22! 0.3.4218 0. that the length of a certain micro RNA is 22.4218 0. 22.1423. U. The event that our microRNA contains 14 purines can be represented by X = 14. the probability that the number of Heads is lower than or equal to two P (X ≤ 2) is computed by pbinom(2.6.1406 0.9860. 14!(22 − 14)! This is the value of the density function at 14.7. That is. Suppose. DISCRETE DISTRIBUTIONS > for (k in 0:3) print(dbinom(k. From the table we read that the probability of no albino child is 0. 22. for the purpose of illustration. and that the process of placing purines and pyrimidines is binomially distributed.7) = 0. Table 3. G.9843 1 Example 3.1.0156.1865.3. and C. that the probability of a purine equals 0. RNA consists of a sequence of nucleotides A. The probability of strictly more than 10 purines is 22 P (X ≥ 11) = k=11 P (S22 = k) = sum(dbinom(11 : 22. that is P (X ≤ 13) = pbinom(13. The values of the density and distribution function are summarized in Table 3.1. number of Heads k=0 k=1 k=2 k=3 density P (X = k) 0.0.25). 22. 0.7)) = 0.0156 distribution P (X ≤ k) 0.3.843 0. where the ﬁrst two are purines and the last two are pyrimidines.4218 and the probability that all three children are albino equals 0.25)) 33 Changing d into p yields the so-called distribution function with the cumulative probabilities.7) = 0.1: Discrete density and distribution function values of S3 . 0.714 0. The binomial density function can be plotted by: .4218 0. The probability of the event of less than or equal to 13 purines equals the value of the distribution function at value 13. 0.0.

00 x 0 5 10 15 20 0 5 10 15 20 0.1: Binomial probabilities with n = 22 and p = 0.0 0.7 and length 22.7).0:22 > plot(x.34 CHAPTER 3. 2.0 x Figure 3.size=22.2 illustrates that the distribution is an increasing step function.7 can be drawn by the command rbinom(1000. · · · . F.7). explained and illustrated. The graph in Figure 3.0. This simulates the number of purines in 1000 microRNA’s each with purine probability equal to 0. .dbinom(x. and chi-squared will be deﬁned.2 0. From Figure 3.10 0. > x <. with x on the horizontal axis and P (X ≤ x) on the vertical.22.6 0.4.8 F(x) 1.2: Binomial cumulative probabilities with n = 22 and p = 0.prob=.7. 3.1 it can be observed that the largest probabilities occur near the expectation 15. where the argument h speciﬁes pins.2 Continuous distributions The continuous distributions normal.7 Figure 3. A random sample of size 1000 from the binomial distribution with n = 22 and p = 0. 22} is constructed and by the second the density function is plotted.4 0.05 0. IMPORTANT DISTRIBUTIONS f(x) 0.15 0. T.type="h") By the ﬁrst line the sequence of integers {1.

2. 0.4) = 1 − pnorm(2. That is. 0.90. From the graph of its density function in Figure 3. the probability of the population to have values smaller than or equal to x.normal() to launch an interactive display of bell-shaped curves. The value of the distribution function is given by P (X ≤ x).4. Figure 3. then the curves moves to zero so that extreme values occur with small probability.9. xn are seen as realizations of a random variable X having a normal distribution. when the mean µ increases.52 ). Example 2. · · · .4) = pnorm(1. then the distribution is steep/ﬂat.90. To view members of the normal distribution load the TeachingDemos package and give the command vis. σ 2 ) for the normal distribution.4 illustrates the value 0.1586. 1. the data values x1 . A density function may very well be seen as a histogram with arbitrarily small bars (intervals). . It corresponds to the area of the blue colored surface below the graph of the density function in Figure 3.2. Suppose that the expression values of gene CCND3 Cyclin D3 can be represented by X which is distributed as N (1. If σ is small/large.4.1586.3.3. Example 1.3.9. Equivalently one says that the data values are members of a normally distributed population with mean µ (mu) and variance σ 2 (sigma squared). 1.5) = 0. The probability that the expression values are larger than 2. then the distribution moves to the right. 0. The probability that the expression values are less then 1. it can be observed that it is symmetric and bell-shaped around µ = 1.4 is P (X ≥ 2.4 is P (X < 1. The curves are symmetric around µ and attain a unique maximum at x = µ. Various properties of the normal distribution are illustrated by the examples below. In particular. If x moves further away from the mean µ. It is good custom to use Greek letters for population properties and N (µ.4.1 Normal distribution The normal distribution is of key importance because it is assumed for many (preprocessed) gene expression values. These bell-shaped curves are also called normal densities. Move the Mean and the Standard Deviation from the left to the right to explore their eﬀect on the shape of the normal distribution.5) = 0.16 of the distribution function at x = 1. CONTINUOUS DISTRIBUTIONS 35 3.

rnorm(1000.4 0. the quantile x0.920018.9.1.8862 and sd(x)=0.4 2 x 3 4 Figure 3.5071 are close to their population values µ = 1.4. Hence.4 equals P (1. 2 2 Use the function round to print the mean in a desired number a decimal places.920018 That is.025. When X is distributed as N (1. then the population mean is 1.8 0.6 normal distribution F(x) density f(x) 0.9.90.9 and σ = 0. The probability that X is between 1. The graph of the distribution function in Figure 3. 1.9. Figure 3.4 illustrates that it is strictly increasing.4 and 2.4 0 1 2 x 3 4 0 1 0.2 0.9 and standard deviation 0.5) The estimate mean(x)=1.4 ≤ X ≤ 2. it holds that the probability of values smaller than 0.16 1.5.920018 equals 0.0 1.0 0.4.8 1.025 can be computed by > qnorm(0.0.1.920018) = 0.5.5) [1] 0. 1. 1.5.5).4 P(X<=1. 0.3: Graph of normal density with mean 1.920018.025.9 and the population standard deviation 0.16 0. 0.36 CHAPTER 3.5) − pnorm(1.0.4) = 0.9. The exact value for the quantile x0.4: Graph of normal distribution with mean 1. IMPORTANT DISTRIBUTIONS 0. . 0. 0.9. To verify this we draw a random sample of size 1000 from this population by > x <.025.025 = 0. that is P (X ≤ 0.0 0.5.9545. as can be veriﬁed by pnorm(0.52 ).6 0.5) = 0.4) = pnorm(2.9 and standard deviation 0.2 0.

Let’s consider the chi-squared variable with 5 degrees of 2 2 freedom.025 . ”Visualizing the Chi-squared”. and adapt ”Xmax”. it holds that (X − µ)/σ = Z is distributed as N (0. 3.6). Then the sum of squares m 2 2 χ2 = Z1 + · · · + Zm = m i=1 Zi2 . Often we are interested in the value for the quantile x0. let {Z1 .2. Example 1. as follows. This value corresponds to the area of the blue colored surface below the graph of the density function in Figure 3. σ 2 ). χ2 = Z1 + · · · + Z5 . To view various members of the χ2 distribution load the TeachingDemos package. 5) = 0. 5 This yields the value of the distribution function at x = 8 (see Figure 3. To compute the probability of values smaller 5 than eight we use the function pchisq.2 Chi-squared distribution The chi-squared distribution plays an important role in testing hypotheses about frequencies. Zm } be independent and standard normally distributed random variables. then there exists an exact and unique solution for the quantiles. CONTINUOUS DISTRIBUTIONS 37 For X distributed as N (µ. Thus by subtracting µ and dividing the result with σ any normally distributed variable can be standardized into a standard normally distributed Z having mean zero and standard deviation one. P χ2 ≤ 8 = pchisq(8.5. 3 .025 ) = 0. 1). To deﬁne it. Move the ”Shape” button to the right to increase the degrees of freedom.3.gamma() to open an interactive display of various distributions. Click on ”Visualizing the gamma”. Use the command vis.2. · · · . 3 Such can be computed 5 by If the distribution function is strictly increasing. is the so-called chi-squared distributed (random) variable with m degrees of freedom. Observe that the graphs of chi-squared densities change from heavily skew to the right into more bellshaped normal as the degrees of freedom increases.8437644.025. where P (χ2 ≤ x0. see Chapter 4. Example 2.

4 area=0. lower.gol. data(golub) gol.tail=FALSE) . Then the standardized values are zi = (xi −1. labels= c("ALL".tail=TRUE) [1] 0. Hence.factor(golub.golub[1042. then the sum of squared standardized values equals their number and the probability of larger values is about 1/2.84 1. 0.00 0.84 0. The probability of larger values 1 is P (χ2 ≥ 25. al.0 8 0 5 10 15 20 25 0 5 8 10 15 20 x Figure 3. Using R the computations are as follows.90.2 0. 5 Example 3. With respect to the Golub et.38 CHAPTER 3.0 0.fac <.90)/0.50 and their sum of squares 27 zi2 = 25. 5 > qchisq(0.8312 Figure 3.fac=="ALL"] z <. let x1 .8 0. IMPORTANT DISTRIBUTIONS 0.03312.90)/0. x27 be the gene expression values.15 Chi−Squared Distribution F(x) Chi−Squared Density f(x) 0."AML")) x <.5: χ2 -density. 5. which indicates that this normal distribution 27 ﬁts the data well.10 0.6 0.(x-1. library(multtest).cl.levels=0:1. If this indeed holds.27. The chi-squared distribution is frequently used as a so-called goodness of ﬁt measure.50 sum(z^2) pchisq(sum(z^2).05 0. In particular.025. · · · .03312) = 0.5726. it is likely that the speciﬁed normal distribution is indeed correct.502 ). (1999) data we may hypothesize that the expression values of gene CCND3 Cyclin D3 for the ALL patients are distributed as N (1.6: χ2 distribution. lower.

For this reason we take µ = 0.0 −4 −2 0 x−axis 2 4 Figure 3.3.2 0. The expression values of this gene are collected in row 2058 of the √ golub data.2. in particular when the sample size √ lower is than thirty. To compute the sample t-value n(x − µ)/s use n <.11 x <. Figure 3. 0.1 0.2. CONTINUOUS DISTRIBUTIONS 39 3.4 0.fac=="AML"] .t() to explore a visualization of the T -distribution.2 0. A quick NCBI scan makes it reasonable to assume that the gene Gdf5 has no direct relation with leukemia.3 T-Distribution The T -distribution has many useful applications for testing hypotheses about means of gene expression values. Load the TeachingDemos and give vis. Example 2.0 0.0 0. Example 1. then the values of n(x − µ)/s follow a T -distribution with n−1 degrees of freedom. gol.8: Distribution function of T10 .7: Density of T10 distribution. If the data are normally distributed.3 Distribution F(x) −4 −2 0 x−axis 2 4 Density f(x) 0.6 0. The T -distribution is approximately equal to the normal distribution when the sample size is thirty.4 0.8 1. Click on ”Show Normal Distribution” and increase the number of degrees of freedom to verify that df equal to thirty is suﬃcient for the normal approximation to be quite precise.golub[2058.

we shall .gol.sqrt(n)*(mean(x)-0)/sd(x) t. 10) − pt(−2. s2 that of the second.8. 10) = 0. P (T10 ≥ 1.236324) = 1 − pt(1.n-1)=-2.025. With respect to the Golub et. This probability corresponds to the area of the blue colored surface below of the graph of the density function in Figure 3. The T distribution function with ten degrees of freedom is illustrated in Figure 3. where s2 is the 1 variance of the ﬁrst set. More speciﬁ2 2 cally. The 2.236324 can be computed. 10) = 0.236324 From the above we know that this has a T10 distribution.7. The probability that the random variable T10 is between -2 and 2 equals P (−2 ≤ T11 ≤ 2) = pt(2.12. not border. (1999) data it is easy to compute the ratio of the variances of the expression values of gene CCND3 Cyclin D3 for the ALL patients and the AML patients.fac=="AML"]) [1] 0.2.236324) = 1 − P (T10 ≤ 1. n2 − 1 degrees of freedom.4 Example 1. 3. and n1 is the number of ob2 servations in the ﬁrst and n2 in the second.value [1] 1.4 F-Distribution The F -distribution is important for testing the equality of two variances.gol. The probability that T10 is greater than 1.7116441 2 2 2 2 It is more correct to deﬁne S1 /S2 for certain random variables S1 and S2 .228139. 4 . It can be shown that the ratio of variances from two independent sets of normally distributed random variables follows an F -distribution.value <. IMPORTANT DISTRIBUTIONS t. as follows. then s2 /s2 follows 1 2 an F -distribution with n1 − 1.fac=="ALL"])/var(golub[1042.926612.236324. > var(golub[1042. al. however. if the two population variances are equal (σ1 = σ2 ). For equal population variances the probability is large that that the ratio of sample variances is near one.40 CHAPTER 3.5% quantile can be computed by qt(0.

0 0.0.025 use qf(. as follows.10: Distribution of F26. This subject is taken further in Section 4.5.2 0.3.7116441. Figure 3. Figure 3.23 0.10 gives the distribution function. Figure 3.9: Density of F26.9.1.0 0. > f<-function(x){dnorm(x.0.7116441) = pf(0.5 5 Plotting a density function A convenient manner to plot a density function in by using the corresponding built-in-function.6 F Distribution F(x) F density f(x) 0.23 0.10 . the probability that the ratio attains values smaller than 0.8 1.7116441 is P (X ≤ 0. CONTINUOUS DISTRIBUTIONS 41 0.26.2326147.ylab="density f(x)") 5 This subsection is solemly on plotting and can be skipped without loss of continuity. 10) = 0.71 0 2 4 6 8 10 Figure 3.10 distribution.2.10)=0.025.9 illustrates that this value corresponds to the area of the blue colored surface below the graph of the density function.xlab="x-axis".2. Since n1 = 27 and n2 = 11 this ratio is a realization of the F26.3861673.8 0. To ﬁnd the quantile x0.10 . 3.4.0 0.71 0 2 4 6 8 10 x 0.6 0.4 0. . For instance to plot the bell-shaped density from the normally distributed variable use the function dnorm.5)} > plot(f. 26. Then.2 0.4 0.1.

m. σ) Chi-squared m dchisq(x. σ) rnorm(10. normal. The speciﬁcation 0.2: Built-in-functions for random variables used in this chapter. n. beta. m.3. µ. m) rchisq(10. or Dirichlet. m) qchisq(α. p for (cumulative) probability distribution.0.0). The freeware encyclopedia wikipedia often gives a useful ﬁrst. χ2 -distributions. there are several additional distributions available such as the Poisson. n. In particular.1. m) pchisq(x. p) pbinom(x.x. where d stands for density.1. σ dnorm(x. q for quantiles. µ. the command polygon is used to give the surface below the graph the color "lightblue". c(0. Gamma. n. p dbinom(x. Obviously. p) Normal µ.ylab="density f(x)") x<-seq(0.2. µ.0. The density. Table 3. col="lightblue") The basic idea of plotting is to start with a plot and next to add colors. expectation. m) F m. pararandom Distribution meters density distribution quantiles sampling Bin n.4 deﬁnes the interval on the horizontal axis over which f is plotted. We can give the surface under f running x from 0 to 1. The vertical axis is adapted automatically. σ) pnorm(x. see Table 3. m) rt(10. m) qt(α. m. n) pf(x. m) pt(x.3. orientation. 3.4 a nice blue color by using the following. m. Note that a distribution acts as a population from which a sample can be drawn. µ. n) qf(α.4). etc. n) rf(10. The polygon (surface enclosed by many angles) is deﬁned by the sequence of points deﬁned as x and f(x). these can also be programmed by yourself. IMPORTANT DISTRIBUTIONS This produces the graph of the density function in Figure 3. n) Although for a ﬁrst introduction the above distributions are without doubt among the most important. t. and variance of most the distributions in this chapter are summarized in Table 3. and r for drawing random samples. though technical.01) polygon(c(0. p) qbinom(α.3 Overview and concluding remarks For practical computations R has built-in-functions for the binomial. σ) qnorm (α.f(x). F.xlab="x-axis". p) rbinom(10.4.42 CHAPTER 3. m) T m dt(x.n df(x.4. n. plot(f. Hence. arrows. distributions . text.

4. (f) The quantiles z0.3).975 .3.5 . (c) P (20 ≤ X or X ≥ 40). EXERCISES 43 can be seen as models of data generating procedures. z0. and x0. 1. 2) the following probabilities and quantiles. Compute the following.4. (b) P (20 ≤ X ≤ 30). Binomial Let X be binomially distributed with n = 60 and p = 0. P (20 ≤ X).3: Density. (c) P (−1. Normal. (b) P (Z < 1. Distribution parameters density expectation variance n! Binomial n.64 < Z < −1. Standard Normal.96). P (X ≤ 24). (e) The quantiles x0.64). . For a more thorough treatment of distribution we refer the reader to Bain & Engelhardt (1992). and P (X ≥ 30). Compute for X distributed as N (10. x0. (a) P (X = 24). Compute the following probabilities and quantiles.05 . p np np(1 − p) pk (1 − p)n−k k!(n−k)! 1 1 x−µ 2 √ exp(− 2 ( σ ) ) Normal µ.02).975 .4 Exercises It is importance to obtain some routine with the computation of probabilities and quantiles. σ µ σ2 σ 2π Chi-squared df=m m 2m 3. z0. z0. Johnson et al. (d) P (0 < Z < 1. 3. Table 3. (a) P (1. (d) Compute the mean and standard deviation of X. mean.5 .96). and variance of distributions used in this chapter.96 < Z < 1.6 < Z < 2. (e) P (−1.95 . and Miller & Miller (1999).025 . 2. (1992).025 . and z0. and P (20 ≤ X and X ≥ 10).

and x0. (b) P (T6 > 2).025 . (d) The quantiles f0.025 . (e) The quantiles t0. (c) P (1 < F8. (d) P (−2 < T6 < −2).975 . MicroRNA. (d) The quantiles x0. 10 (b) P (χ2 > 4). and t0.5 < 6). and g0. and f0. IMPORTANT DISTRIBUTIONS (c) P (9 < X < 10. x0. Verify the following computations for the T6 distribution. (b) P (X > 8). 4. (b) P (F8. 7.5 < 3).44 (a) P (X < 12). g0.5 .5 . (a) P (χ2 < 3).5 distribution. CHAPTER 3.7. (a) P (T6 < 1).975 .5 .025 . F distribution.975 . 5).975 .5 > 4). (a) What is the probability of 14 purines? (b) What is the probability of less than or equal to 14 purines? . 5. Compute the following probabilities and quantiles for the F8.5 . f0. 10 (d) The quantiles g0. (c) P (−1 < T6 < 1). (a) P (F8. 6. Compute the following for the chi-squared distribution with 10 degrees of freedom. Chi-squared distribution. 10 (c) P (1 < χ2 < 6).025 . T -distribution. Suppose that for certain microRNA of size 20 the probability of a purine is binomially distributed with probability 0. t0.

9.975 . How many are between 0. subtract from these maxima an and divide by bn.5? 10. (a) Compute the probability that the expression values are smaller than 1.2? (b) What is the probability that the expression values are between 1.0? (c) What is the probability that the expression values are between 0. The distribution of the expression values of the ALL patients on the Zyxin gene are distributed according to N (1. (e) Use rnorm to draw a sample of size 1000 from the population and compare the sample mean and standard deviation with that of the population. p. Find the three genes with largest absolute t-values.(2*log(n))^(-1/2) . So that you sampled 1000 maxima.5 and 1.6. 0.8 and 2.3. Some computations on Golub et al.4.103).025 and x0. (a) Take µ = 0 and compute the t-values for the ALL gene expression values. (1999) data. Take the maximum of a sample (with size 1000) from the standard normal distribution and repeat this 1000 times.4? (d) Compute the exact values for the quantiles x0. This (diﬃcult!) question aims to teach the essence of an extreme value distribution! An interesting extreme value distribution is given by Pevsner (2003. Zyxin.5*(log(log(n))+log(4*pi))*(2*log(n))^(-1/2) bn <.sqrt(2*log(n)) .0.2 and 2. where an <.42 ). EXERCISES (c) What is the probability of strictly more than 10 purines? 45 (d) By what probability is of the number of purines between 10 and 15? (e) How many purines do you expect? In other words: What is the mean of the distribution? (f) What is the standard deviation of the distribution? 8. (b) Compute per gene the ratio of the variances for the ALL and the AML patients. Next. Extreme value investigation.

What do you observe? . and add the density (dnorm) from the normal distribution. IMPORTANT DISTRIBUTIONS Now plot the density from the normalized maxima and add the extreme value function f (x) from Pevsner his book.46 CHAPTER 3.

Once estimates are available it becomes possible to statistically test biologically important hypotheses. The current chapter gives several basic examples of statistical testing and some of its background. With 47 . In any research (empirical) setting the speciﬁc values of such parameters are unknown so that these must be estimated. Does the mean gene expression over ALL patients diﬀer from that over AML patients? That is.1 Statistical hypothesis testing Let µ0 be a number representing the hypothesized population mean by a researcher on the basis of experience and knowledge from the ﬁeld. 4.Chapter 4 Estimation and Inference Questions that we deal with in this chapter are related to statistically testing biological hypothesis. does the mean gene expression level diﬀer between experimental conditions? Is the mean gene expression diﬀerent from zero? To what extent are gene expression values normally distributed? Are there outliers among a sample of gene expression values? How can an experimental eﬀect be deﬁned? How can genes be selected with respect to an experimental eﬀect? Other important questions are: How can it be tested whether the frequencies of nucleotide sequences of two genes are diﬀerent? How can it be tested whether outliers are present in the data? What is the probability of a certain micro RNA to have more than a certain number of purines? In the foregoing chapters many population parameters were used to deﬁne families of theoretical distributions. Robust type of testing is brieﬂy introduced as well as an outlier test.

The corresponding expression values are contained in row 2058.1 The Z-test The Z-test applies to the situation where we want to test H0 : µ = µ0 against H1 : µ = µ0 and the standard deviation σ is known.fac.1. 4. so it is essential to load these data and to deﬁne the factor gol.1 Accordingly. then H0 is not rejected and if it is smaller than the signiﬁcance level. After conducting the experiment.48 CHAPTER 4. A quick search through the NCBI site Recall from a calculus course that | − 2| = 2 and |2| = 2. the value of the statistic can be computed from the data. We will work with golub throughout this chapter. 2 1 . · · · . standardized mean). xn ) are from a normal distribution we compute √ the standardized value z = n(x − µ0 )/σ. is called the signiﬁcance level which is generally denoted by α. The probability to reject H0 . We shall follow the habit in statistics to use α = 0.05. The alternative hypothesis is true if H1 : µ < µ0 or H1 : µ > µ0 holds true. that is occurring to the left of −|z| or to the right of |z|. Next we deﬁne the so-called pvalue as the standard normal probability of Z attaining values being more extreme than |z|. the researcher draws a conclusion with respect to the null hypothesis: H0 is rejected or it is not. To illustrate the Z-test we shall concentrate on the Gdf5 gene from the Golub et al. ESTIMATION AND INFERENCE respect to the population mean the null hypothesis can be formulated as H0 : µ = µ0 and the alternative hypothesis as H1 : µ = µ0 . but it will be completely clear how to adapt the procedure in case other signiﬁcance levels are desired. the p-value equals P (Z ≤ −|z|) + P (Z ≥ |z|) = 2 · P (Z ≤ −|z|). Such a null hypothesis will be statistically tested against the alternative using a suitable distribution of a statistic (e.g. This type of alternative hypothesis is called “two-sided”. In case H1 : µ > µ0 . it is called “one-sided”. Assuming that the gene expression values (x1 . By comparing the value of the statistic with its distribution. These are two statements of which the latter is the opposite of the ﬁrst: Either H0 or H1 is true. given the truth of H0 . (1999) data2 . Example 1. then H0 is rejected. The conclusion from the test is now as follows: If the p-value is larger than the signiﬁcance level α.

3 If we would repeat the procedure suﬃciently often .sqrt(n)*(mean(x) . we are 95% certain3 that the true mean falls in the conﬁdence interval. the 95% conﬁdence interval for the population mean µ is σ σ x + z0.0. z0. > 2*pnorm(-abs(z. labels= c("ALL".0 x <.05.0. we conclude that the null hypothesis of mean equal to zero is not rejected (accepted). The interval (z0.27. ∞).gol. if z falls in the region (−∞.025 . In particular. For this reason these intervals are called “rejection regions”. x + z0.001116211) can be computed as follows.value).25. n <. Such an interval is standard output of statistical software.975 . Hence. then H0 is not rejected and consequently this region is called ”acceptance region”.factor(golub.25.025 ] or [z0.1. Accordingly. The situation is illustrated in Figure 4.1.levels=0:1. mu0 <.025 √ .fac=="ALL"] z.1) That is. we test H0 : µ = 0 against H1 : µ = 0. z0."AML")) sigma <.975 ) is often named “conﬁdence interval”.975 √ n n . p. For the sake of illustration we shall pretend that the standard deviation σ is known to be equal to 0.025 . then we are 95% conﬁdent that the observed z-value falls in it.golub[2058. z0.1) [1] 0. Note that the above procedure implies rejection of the null hypothesis when z is highly negative or highly positive. we may hypothesize that the population mean of the ALL expression values equals zero. data(golub. because if the null hypothesis is true. (4.value <. It is custom to rework the conﬁdence interval into an interval with respect to µ (Samuels & Witmer. More precisely. STATISTICAL HYPOTHESIS TESTING 49 makes it likely that this gene is not directly related to leukemia. 186). 2003.cl. then H0 is rejected.fac <.4.9991094 Since it is clearly larger than 0. The z-value (=0.975 ). If z falls in the interval (z0.mu0)/sigma The p-value can now be computed as follows. package = "multtest") gol.

Using the data from Example 1. the rounded estimated 95% conﬁdence interval is (−0.50 CHAPTER 4.0.3 0. 4 . It is instructive and convenient to run the Z-test from the TeachingDemos package.1)*sigma/sqrt(n) [1] 0.0942451 > mean(x)+qnorm(c(0.2 area α 2 0.094. H0 is not rejected. especially the deﬁnition of x.1)*sigma/sqrt(n) [1] -0.1 acceptance area 1 −α rejection area α 2 0. as follows.975). Since µ0 = 0 falls within this interval. These computations only work together with those of Example 1. ESTIMATION AND INFERENCE normal density 0.4 rejection 0. 0.0 z −4 −2 0 2 4 Figure 4.094). Example 2.025). the 95% conﬁdence interval given by Equation 4.1 can be computed as follows.1: Acceptance and rejection regions of the Z-test.4 > mean(x)+qnorm(c(0.09435251 Hence.0.

p-value = 0.4.025.975) Then 100 samples of size 25 from the N (0.examp(mean.09424511 0. Dev. of the sample mean = 0. This illustrates that testing by either of these procedures yields equivalent conclusions. Std. Dev. 1) distribution are drawn and for each of these the conﬁdence interval for the population mean is computed and represented as a line segment. STATISTICAL HYPOTHESIS TESTING > library(TeachingDemos) > z. 4.048. the p-value. upper. reps = 100.1. Example 3.250.0011. To develop intuition with respect to conﬁdence intervals load the package TeachingDemos and give the following command. n = 27. sd = 1.1.25) One Sample z-test 51 data: x z = 0.test(x. Apart from sampling ﬂuctuations. > + ci. the conclusion is not to reject the null-hypothesis of mean equal to zero.2 One Sample t-Test Indeed. in almost all research situations with respect to gene expression values.9991 alternative hypothesis: true mean is not equal to 0 95 percent confidence interval: -0. lower. = 0.conf=0. the population standard deviation σ is unknown so that the above test is not applicable. Std. In such cases t-tests are very useful for testing H0 : µ = µ0 against H1 : µ = µ0 . n = 25.conf=0.sd=0. The test is based on the t-value deﬁned by t = .09435251 sample estimates: mean of x 5. and the conﬁdence interval.000.mu=0.sim =0.37037e-05 From the z-value. the conﬁdence level corresponds to the percentage of intervals containing the true mean (colored in black) and that the signiﬁcance level corresponds to intervals not containing it (colored in red or blue). method = "z".

025.975 4 Figure 4. Let’s test H0 : µ = 0 against H1 : µ = 0 for the ALL population mean of the Gdf5 gene expressions.2 region α 2 acceptance region rejection region α 2 0. x + t0.2: Acceptance and rejection regions of the T5 -test.3 T density rejection 0.1 t0.n−1 . if t falls in the acceptance region (t0.n−1 ). Example 1.0 0. Similar to the above. For n = 6 the acceptance and rejection regions are illustrated in Figure 4.025 −4 −2 0 x−axis 2 t0. The latter are collected in row 2058 of the golub data.52 √ CHAPTER 4. . ESTIMATION AND INFERENCE n(x − µ0 )/s.025 · s/ n. then H0 is not rejected and otherwise it is.2. where √ the expression s/ n gives the so-called “standard error of the mean”. Equivalently.975 · s/ n). The corresponding p-value is deﬁned by 2 · P (Tn−1 ≤ −|t|). The 95% conﬁdence interval √ √ for the population mean is given by (x + t0.975. H0 is not rejected if the p-value is larger than the significance level and H0 is rejected if the p-value is smaller than the signiﬁcance level. t0. 0. The t-value is computed as follows.

gol. 26) = 0. p-value = 0. Since this interval does contain the t-value.0.025.27 > t.1.golub[2058.26 ) = (qt(0.fac=="ALL"].26 .975. > mean(x)+qt(0. as follows.025. 53 so that the conclusion is not to reject the null hypothesis of mean equal to zero. n − 1).0010. n − 1)) = (−2. we compute (t0.055). The left boundary of the 95% conﬁdence interval for the population mean can be computed.9991 > α. qt(0.1025.1024562 The 95% conﬁdence interval equals (−0.0011.37037e-05 .test(x.test. In daily practice it is much more convenient to use the built-in-function t. STATISTICAL HYPOTHESIS TESTING > x <. 2. Since it contains zero. To see whether the observed t-value belongs to the 95% conﬁdence interval.975.value [1] 0.9991 alternative hypothesis: true mean is not equal to 0 95 percent confidence interval: -0.055.26)*sd(x)/sqrt(n) [1] -0.1024562 0.001076867 The corresponding p-value can be computed by 2 · P (T26 ≤ −0. n <. We illustrate it with the current testing problem. we do not reject the hypothesis that µ equals zero. df = 26.value<-sqrt(n)*(mean(x) .025. 0. mu0 <.1025).mu0)/sd(x) > t. t0.4.mu=0) One Sample t-test data: x t = 0. we do not reject the null-hypothesis.1025636 sample estimates: mean of x 5. > t.0010) = 2 ∗ pt(−0.

alternative = c("greater")) One Sample t-test data: golub[1042. p-value < 2. Recall that the corresponding gene expression values are collected in row 1042 of the golub data matrix (load it if necessary). then the alternative hypothesis is one-sided and this makes the procedure slightly diﬀerent: H0 is accepted if P (Tn ≥ t) > α and it is rejected if P (Tn ≥ t) < α.gol. so that the conclusion is to reject H0 . the mean differs largely from zero.2e-16 alternative hypothesis: true mean is greater than 0 95 percent confidence interval: 1.54 CHAPTER 4. Accordingly.fac == "ALL"] t = 20. Example 2.fac=="ALL"].1. H0 : µ1 = µ2 is to . gol. and the 95% conﬁdence interval for µ0 . In particular.0599. We shall illustrate this by a variant of the previous example.test(golub[1042.mu=0. the researcher desires to test H0 : µ = µ0 against H1 : µ > µ0 . If. df = 26. Hence. the p-value. In the previous example the test is two-sided because H1 holds true if µ < µ0 or µ > µ0 . however.3 Two-sample t-test with unequal variances Suppose that gene expression data from two groups of patients (experimental conditions) are available and that the hypothesis is about the diﬀerence between the population means µ1 and µ2 . we test H0 : µ = 0 against H1 : µ > 0 by the built-in-function t-test. 4. the p-value is very close to zero. In Chapter 2 a box-and-whiskers plot revealed that the ALL gene expression values of CCND3 Cyclin D3 are positive. ESTIMATION AND INFERENCE This yields by one command line the observed t-value. relative to its standard error. > t.893883 The large t-value indicates that.732853 Inf sample estimates: mean of x 1.

df = 16. > t.test and the appropriate factor and speciﬁcation var. Let x be the mean of the ﬁrst and y that of the second. p-value = 9. · · · xn } and that of the second by {y1 .fac t = 6.] ~ gol. the conclusion is to reject the null-hypothesis of equal means.3186.118. ym }. var. the standard deviations s1 and s2 are small.87e-06 alternative hypothesis: true difference in means is not equal to 0 95 percent confidence interval: 0. indicating that the two means x and y diﬀer largely from zero relative to the corresponding standard error (denominator in Equation 4. Then the t-statistic can 2 be formulated as (x − y) − (µ1 − µ2 ) t= .2) s2 /n + s2 /m 1 2 The decision procedure with respect to the null-hypothesis is completely similar to the above tests.equal=FALSE) Welch Two Sample t-test data: golub[1042.4. The boxplot in Figure 2. ] by gol. (1999) argue that gene CCND3 Cyclin D3 plays an important role with respect to discriminating ALL from AML patients. The data provide strong evidence that the 5 Assuming µ1 − µ2 = 0. This test is known as the Welch two-sample t-test (Lehmann. and s2 1 the variance of the ﬁrst and s2 that of the second. These hypotheses can also be formulated as H0 : µ1 − µ2 = 0 and H1 : µ1 − µ2 = 0. . Since the p-value is extremely small. Suppose that gene expression data from the ﬁrst group are given by {x1 .fac. STATISTICAL HYPOTHESIS TESTING 55 be tested against H1 : µ1 = µ2 . and the sample sizes are large. The null hypothesis of equal means can be tested by the function t. · · · .6802008 sample estimates: mean in group ALL mean in group AML 1.2). Golub et al. Example 1. 1999).8363826 1. Note that the t-value is large if the diﬀerence between x and y is large5 .equal=FALSE. (4.6355909 The t-value is quite large.test(golub[1042.4 suggests that the ALL population mean diﬀers from that of AML.8938826 0.1.

namely 1 2 s2 = p (n − 1)s2 + (m − 1)s2 1 2 . 4. When the ﬁrst group is an experimental group and the second a control group. + Example 1.test(golub[1042.56 CHAPTER 4. then µ1 −µ2 is the experimental eﬀect in the population and x−y that in the sample.equal = TRUE) Two Sample t-test data: golub[1042. The null hypothesis for gene CCND3 Cyclin D3 that the mean of the ALL diﬀers from that of AML patients can be tested by the two-sample t-test using the speciﬁcation var.8829143 1. ] by gol. ESTIMATION AND INFERENCE population means do diﬀer.fac t = 6.4 Two sample t-test with equal variances Suppose exactly the same setting as in the previous paragraph. but now 2 2 the variances σ1 and σ2 for the two groups are known to be equal. If the two population variances are equal. there is a t-test which is based on the so-called pooled sample variance s2 . then the testing procedure simpliﬁes considerably. df = 36. p-value = 6. The latter is deﬁned by the following p weighted sum of the sample variances s2 and s2 .fac. n+m−2 Then the t-value can be formulated as t= x − y − (µ1 − µ2 ) sp 1 n 1 m .7983. The t-value is the experimental eﬀect in the sample relative to the standard error. To test H0 : µ1 = µ2 against H1 : µ1 = µ2 . This is the subject of the next paragraph. > t. The size of the eﬀect is measured by the p-value in the sense that it is smaller for larger eﬀects.6336690 .1. var.046e-08 alternative hypothesis: true difference in means is not equal to 0 95 percent confidence interval: 0.equal=TRUE.] ~ gol.

the conclusion is to reject the null hypothesis of equal population means. If P (Fn1 −1.046 · 10−8 .test(golub[1042. denom df = 10.1.n2 −1 < f ) ≥ α/2 for f < 1 or P (Fn1 −1. The null hypothesis for gene CCND3 Cyclin D3 that the variance of the ALL patients equals that of the AML patients can be tested by the built-in-function var.n2 −1 > f ) ≥ α/2 for f > 1.fac) F test to compare two variances data: golub[1042. In case of any uncertainty about the validity of the assumption of equal population variances. p-value = 0.5 F-test on equal variances The assumption of the above t-test it that the two population variances are equal. one may want to test this.6355909 57 From the p-value 6. as follows.8938826 0.4652 alternative hypothesis: true ratio of variances is not equal to 1 95 percent confidence interval: 0. which is Fn1 −1.7116441 . Note that the p-value is slightly smaller than that of the previous test.7116. num df = 26. 4. ] by gol. Such an assumption can serve as a null hypothesis.4. then H0 is not rejected and otherwise it is rejected.8428387 sample estimates: ratio of variances 0. From the sample variances s2 and s2 . Example 1. That is.fac F = 0. we desire 2 2 2 2 to test H0 : σ1 = σ2 against H0 : σ1 = σ2 .2127735 1.test.] ~ gol. as follows.n2 −1 distributed with 1 2 n1 − 1 and n2 − 1 degrees of freedom.1. STATISTICAL HYPOTHESIS TESTING sample estimates: mean in group ALL mean in group AML 1. the 1 2 f -value f = s2 /s2 can be computed. This can be accomplished by the so-called F -test. > var.

4652. Assuming that the binomial distribution holds. p = 0. This test can also be conducted by the function binom.1645 is larger than the signiﬁcance level 0. Example 1. the conclusion follows not to reject the null-hypothesis.95) Exact binomial test data: 18 and 22 number of successes = 18. so that the null hypothesis is not rejected.7 is to be tested against the one-sided H1 : p > 0. number of trials = 22. The null hypothesis H0 : p = 0. > binom. . alternative = c("greater"). the null-hypothesis of equal variances is not rejected.0000000 sample estimates: probability of success 0.7) = 0. A micro RNA of length 22 contains 18 purines. Suppose that sequencing reveals that the micro RNA has k purines out of a total n. However.7 95 percent confidence interval: 0.1645 ≥ 0.6 Binomial test Suppose that for a certain micro RNA a researcher wants to test the hypothesis that the probability of a purine equals a certain value p0 .1645 alternative hypothesis: true probability of success is greater than 0.05. 4. ESTIMATION AND INFERENCE From the p-value 0.test as follows. 22.7.05. p-value = 0. In such a setting we want to test the null-hypothesis H0 : p = p0 against the one-sided alternative hypothesis H1 : p > p0 . then H0 is not rejected and otherwise it is. the null-hypothesis can be tested by computing the p-value P (X ≥ k). 22.7. 0.58 CHAPTER 4. another researcher has reason to believe that this probability is larger. If it is larger than the signiﬁcance level α = 0.level = 0.05 = α.1. + conf.test(18.8181818 The p-value 0.6309089 1. From P (X ≥ 18) = 1 − pbinom(17.

3: Rejection region of χ2 -test.4. 3 . pm ). The p-value m−1 of the chi-squared test is deﬁned as P (χ2 m−1 ≥ q). where i=1 oi is the i-th observed and ei the i-th expected frequency.00 0. · · · . That is. Chi−Squared Density f(x) 0. · · · . then the null hypothesis is not rejected. By multiplying the probabilities with the total number of observations we obtain the expected number of observations (ei = n · pi ). Now we can compute the statistic q = m (oi − ei )2 /ei . If it is larger than the signiﬁcance level. where p1 to pm are given numbers corresponding to the hypothesis of a researcher.10 acceptance region 0.1. · · · .20 0.1.8 0 5 10 15 20 25 q Figure 4. pm ) against H1 : (π1 .05 rejection region 7. · · · .7 Chi-squared test It often happens that we want to test a hypothesis with respect to more than one probability.15 0. πm ) = (p1 . πm ) = (p1 . STATISTICAL HYPOTHESIS TESTING 59 4. and otherwise it is.25 0. the H0 : (π1 . This statistic is chi-squared (χ2 ) distributed with m − 1 degrees of freedom.

π3 .0674.as.5)2 (573 − 541. ESTIMATION AND INFERENCE Example 1. π3 . G.0674 obviously falls far into the right hand side of the rejection region.1" is downloaded. T } to occur in the sequence be given by (π1 . as follows. for the sequence ”X94991.5 541.2e-16 The package ape is loaded. π4 ). The observed frequencies are given as input to chisq. 1/4. df = 3.character=TRUE)) > chisq.0674) is close to zero. so that the corresponding p-value is very close to zero. so that the expected frequencies ei are equal to 2166/4 = 541.1").1” from Table 1. ∞).5 541. and the frequency table is constructed.60 CHAPTER 4. In the year 1866 Mendel observed in large number of experiments frequencies of characteristics of diﬀerent kinds of seed and their oﬀspring.5)2 (394 − 541. The observed q = 187. C. π4 ) = (1/4. where the red colored surface corresponds to the rejection region (7. Remember from the previous chapter that the left bound of this rejection interval can by found by qchisq(0. Example 2. the q-value equals 4 (oi − ei )2 /ei = i=1 (410 − 541. From the corresponding p-value. Let the probability of {A. π2 .95.5)2 (789 − 541. this yielded the frequencies 5474.81. the conclusion is to reject the null hypothesis of equal probabilities. 3).table(read. p-value < 2.5 541. The testing situation is illustrated in Figure 4.1 the total number of nucleotides is n = 2166. the null hypothesis is clearly rejected. P (χ2 [3] ≥ 187. the Zyxin sequence "X94991. A more direct manner to perform the test is by using the built-in-function chisq. Suppose we want to test the hypothesis that the nucleotides of Zyxin have equal probability. π2 .test which has equal probabilities as the default option.test(zyxinfreq) Chi-squared test for given probabilities data: zyxinfreq X-squared = 187. Then the null hypothesis to be tested is (π1 .GenBank(c("X94991.5)2 + + + = 187. In particular. The q-value equals X-squared and the degrees of freedom df = 3.test. The nucleotides of Zyxin do not occur with equal probability.5.5 Since. In particular. 1850 the seed shape . 1/4. Then.0674 541. 1/4).3. > library(ape) > zyxinfreq <.

25. To further illustrate the great ﬂexibility of the chi-squared test another example is given. 0. The null hypothesis of independence.50. can be tested by a chi-square test. A crossing of B and b yields oﬀ spring BB.2. To test the null hypothesis H0 : (π1 .2629.c(0. true negatives (correctly predicted healthy) and false negatives (incorrectly predicted healty).75. The classiﬁcation yields true positives (correctly predicted disease). > pi <. we use the chi-squared test6 .1. the constant representing the ratio of a circle’s circumference to its diameter.5.75.25). we do not reject the null hypothesis.25) > x <-c(5474. as follows.5). 5 true negatives (tn).25) against H1 : (π1 .test(dat) For the sake of clarity the code is somewhat unelegant in using the symbol pi. 6 . as follows. > dat <. Example 3. we may deﬁne a certain cut oﬀ value and classify e.25 (bb). smaller values to be healthy and larger ones to be infected.byrow=TRUE) > chisq.6081 From the p-value 0.75 (BB and Bb) and 0. For the sake of illustration suppose that among twenty patients there are 5 true positives (tp).0. p=pi) Chi-squared test for given probabilities data: x X-squared = 0. 5 false positives (fp). df = 1. 0. In the worst case the prediction by the cut-oﬀ value is independent of the disease state of the patient. π2 ) = (0.6081.matrix(c(5. Bb and bb with probability 0. and 5 false negatives (fn). STATISTICAL HYPOTHESIS TESTING 61 of ornamental sweet peas. These frequencies can be put is a two-by-two table giving the frequencies on two random variables: the true state of the persons and the predicted state of the persons (by the cut oﬀ value).25. Given certain expression values for a healthy control group and an experimental group with a disease. In such a manner cut-oﬀ values can serve as a diagnostic instrument.g. false positives (incorrectly predicted disease). Since Mendel could not distinguish Bb from BB. his observations theoretically occur with probability 0. 1850) > chisq.test(x.75.5. 0.4. π2 ) = (0. 0. p-value = 0.

matrix(c(8. f22 . ESTIMATION AND INFERENCE Pearson’s Chi-squared test with Yates’ continuity correction data: dat X-squared = 0.02535 Since the p-value is smaller than the signiﬁcance level. Suppose that the number of signiﬁcant onco type of genes in Chromosome 1 is f11 = 100 out of a total of f12 = 2000 and the number of signiﬁcant genes in the whole genome is f21 = 300 out of a total of f22 = 6000. p-value = 0. Suppose that the frequencies . Suppose that for another cutoﬀ value we obtain 8 true positives (tp). 2 false positives (fp). df = 1.2. (f12 .byrow=TRUE) > chisq. > dat <.8). Then testing independence yields the following.2. In a two by two table with frequencies f11 . p-value = 0. A related and frequently applied test in Bioinformatics is the Fisher exact test. and 2 false negatives (fn). df = 1. this test is based on the so-called odds ratio f11 f22 /(f12 f21 ).62 CHAPTER 4.2.test(dat) Pearson’s Chi-squared test with Yates’ continuity correction data: dat X-squared = 5. The null-hypothesis of the Fisher test is that the odds ratio equals 1 and the alternative hypothesis that it diﬀers from 1. signiﬁcant genes 100 300 non-signiﬁcant genes 2000 6000 Chromosome 1 genome Example 4.6547 Since the p-value is larger than the signiﬁcance level. 8 true negatives (tn). the null hypothesis of independence is not rejected. and f21 ). Then the odds ratio equals 100 · 6000/(2000 · 300) = 1 and the number of signiﬁcant oncogenes in Chromosome 1 is exactly proportional to that in the genome.2. the null hypothesis of independence is rejected.

matrix(c(300.byrow=TRUE) > fisher.1. p.3000. STATISTICAL HYPOTHESIS TESTING 63 of signiﬁcant oncogenes for Chromosome 1 equals f11 = 300 out of a total of f12 = 500 and for the genome f21 = 3000 out of f22 = 6000. The hypothesis that the odd ratio equals one can now be tested as follows. Other examples of the Fisher test will be given in Chapter 6.347) and the Anderson-Darling test is based on the distribution of the data (Stephens. > dat <. p. To test the hypothesis that the ALL gene expression values of CCND3 Cyclin D3 from Golub et al.500.fac=="ALL"]) Shapiro-Wilk normality test . the null hypothesis of odds ratio equal to one is rejected.01912 alternative hypothesis: true odds ratio is not equal to 1 95 percent confidence interval: 1.372).029519 1.6000).199960 Since the p-value is smaller than the signiﬁcance level. 1999.396922 sample estimates: odds ratio 1. 1986.4. the Shapiro-Wilk test can be used as follows.test(golub[1042. There are more signiﬁcant oncogenes in Chromosome 1 compared to that in the genome. (1999) are normally distributed. 4. gol. Example 1. The Shapiro-Wilk test is based on the degree of linearity in a Q-Q plot (Lehmann.1.test(dat) Fisher’s Exact Test for Count Data data: dat p-value = 0.2.8 Normality tests Various procedures are available to test the hypothesis that a data set is normally distributed. > shapiro.

05. where the suspect value is included for the computation of the mean x and the standard . > library(nortest) > ad. such a hypothesis can be tested by the Grubbs (1950) test. From the normality tests the conclusion is that the diﬀerences in the left tail are not large enough to reject the null-hypothesis that the CCND3 Cyclin D3 expression values are normally distributed. 4.gol.1683 Hence.64 CHAPTER 4.fac == "ALL"] W = 0. ESTIMATION AND INFERENCE data: golub[1042.1. the null-hypothesis to be tested is that a set of gene expression values does not contain an outlier and the alternative is that it is contaminated with at least one outlier.947.1774 Since the p-value is greater than 0.9 Outliers test When gene expression values are not normally distributed. p-value = 0. p-value = 0. Note that the p-values from both tests are somewhat low. For this reason it is useful to be able to test whether a certain set of gene expression values is contaminated by an outlier or not. Under the assumption that the data are realizations of one and the same distribution. gol.test(golub[1042. This test is based on the statistic g = |suspect value − x|/s. The Anderson-Darling test is part of the nortest package which probably needs to be installed and loaded ﬁrst.5215. the same conclusion is drawn as from the Shapiro-Wilk test.1. The appearance of outliers in gene expression data may inﬂuence the value of a (non-robust) statistic to a large extent. Running the test on our CCND3 Cyclin D3 gene expression values comes down to the following.fac=="ALL"]) Anderson-Darling normality test data: scale(golub[1042. Accordingly. then outliers may appear with large probability.fac == "ALL"]) A = 0.5 based on the Q-Q plot that the distribution resembles the normal. gol. This conﬁrms our observation in Section 2. the conclusion is not to reject the null hypothesis that CCND3 Cyclin D3 expression values follow from a normal distribution.

0183 alternative hypothesis: lowest value 0. STATISTICAL HYPOTHESIS TESTING deviation s.45827 is an outlier Since the p-value is smaller than 0.4. We sustain with a brief description of the basic idea and refer the interested reader to the literature on non-parametric . the t-test is an optimal test for testing H0 : µ1 = µ2 against H1 : µ1 = µ2 (Lehmann.10 Wilcoxon rank test In case the data are normally distributed with equal variance.05. U = 0.1. 1999).1.fac=="ALL"]) Grubbs test for one outlier data: golub[1042. however. In the below we shall concentrate on the two-sample Wilcoxon test because of its relevance to bioinformatics. 1999). as follows. In such cases a robust test based on ranks may be preferred as a useful alternative. the data are not normally distributed due to skewness or otherwise heavy tails.test of the outliers package. This can actually be tested by the function grubbs. Hence.4 we have observed that expression values of gene CCND3 Cyclin D3 may contain outliers with respect to the left tail. extreme outliers indicate that the data are non-normally distributed with large probability.test(golub[1042.6580. > library(outliers) > grubbs. the probability of outliers is small. 65 Example 1. gol. the conclusion is to reject the nullhypothesis of no outliers.9264. If. then this optimality does not hold anymore and there is no guarantee that the signiﬁcance level of the test equals the intended level α (Lehmann. p-value = 0. From Figure 2. 4. In case the data are normally distributed.fac == "ALL"] G = 2. Outliers may lead to such an increase of the standard error that a true experimental eﬀect remains uncovered (false negatives). gol. For this reason rank type of tests are developed for which on beforehand no speciﬁc distributional assumptions need to be made.

test. Such . 2006). we want to analyze a set of thousands of (row) vectors with gene expression values which are collected in a matrix. xm . however. In daily practice. To broaden our view we switch from hypotheses about means to those about distributions. then the sum is small.15e-07 alternative hypothesis: true location shift is not equal to 0 Since the p-value is much smaller than α = 0. the conclusion is to reject the null-hypothesis of equal distributions. For example that the x’s are smaller (or larger) than the y’s. By the two-sample Wilcoxon test the data x1 .2 Application of tests to a whole set gene expression data Various tests are applied in the above to a single vector of gene expressions.fac) Wilcoxon rank sum test data: golub[1042. · · · . 4. The null hypothesis is that both distributions are equal (H0 : F = G) and the alternative that these are not.] ~ gol.test(golub[1042. The distribution of the sum of ranks is known so that a p-value can be computed on the basis of which the null hypothesis is rejected if it is smaller than the signiﬁcance level α. ] by gol. y1 . p-value = 6. · · · . An alternative hypothesis may then be formulated as that the distribution of a ﬁrst group lays to the left of a second. 2006).66 CHAPTER 4.g.05. To set the scene let the gene expression values of the ﬁrst group (x1 to xm ) have distribution F and those of the second group (y1 to yn ) distribution G. as follows. The null hypothesis that the expression values for gene CCND3 Cyclin D3 are equally distributed for the ALL patients and the AML patients can be tested by the built-in-function wilcox. The idea is that if the ranks of x’s are smaller than those of the y’s. > wilcox. yn are ranked and the rank numbers of the x’s are summed to form the statistic W after a certain correction (Lehmann. Lehmann. ESTIMATION AND INFERENCE testing (e. Example 1.fac W = 284.

levels=0:1. Example 2.test(x ~ gol. gol.apply(golub. APPLICATION OF TESTS TO A WHOLE SET GENE EXPRESSION DATA67 can conveniently be accomplished by taking advantage of the fact that R stores the output of a test as an object in such a manner that we can extract information such as p-values.4.levels=0:1. by collecting p-values in a vector we can select genes with large diﬀerences between patient groups.test function and stored in the vector pt.pt)) resul[pw<0. 1.test(x ~ gol.27598 Hence.fac=="ALL"]. 1. according to the Shapiro-Wilk test. labels= c("ALL". labels= c("ALL".value) > sum(sh > 0. This and testing for normality will be illustrated by two examples. function(x) wilcox.apply(golub[.73%. a question one might ask is: What is the percentage of genes that passes a normality test? Such can be computed as follows.package="multtest") > gol.05 & abs(pt-pw)>0. Recall that the smaller the p-value the larger the experimental eﬀect."AML")) pt <. Having a data matrix with gene expression values.fac <.test(x)$p. In case the gene expression data are non-normally distributed the t-test may indicate conclusions diﬀerent from those of the Wilcoxon test.2.2.apply(golub."AML")) > sh <. package = "multtest"). > > > > > > data(golub.fac)$p.fac <.data. For the AML expression values this is 60. Example 1.fac)$p.factor(golub. The logical operator & is used to select genes for which the . 58. function(x) shapiro.cl.gol. It can be concluded that about forty percent of the genes do not pass the normality test.] pw pt 456 0.value) resul <. > data(golub. Diﬀerences between these can be investigated by collecting the p-values from both tests and seeking for the largest diﬀerences. function(x) t.2636088 1509 0.factor(golub. Hence.value) pw <.27% of the ALL gene expression values is normally distributed (in the sense of non-rejection).4427477 The p-value is extracted from the output of the t.03215830 0.04480288 0.cl. 1.05)/nrow(golub) * 100 [1] 58.frame(cbind(pw. the Wicoxon test can be far more eﬃcient than the t-test (Lehmann. The latter are assumed to have a certain suitable distribution which is seen as a statistical model for outcomes of an experiment. Then a statistic is formulated (e. These considerations set the scene for making some recommendations. then the Welch type of t-test provides a general test with good power properties (Ramsey. which means that in the optimal situation where the (gene expression) data are normally distributed. 1980. Statistically. Wilcoxon’s test is only a little worse than the t-test. In case normality does not hold and the sample size per group is at least least four. a t-value) which is treated both as a function of the random variables and as a function of the data values. A large p-value indicates that the model ﬁts the data well and that the assumptions as well as the null-hypothesis are correct with large probability. 4.955. however. the p-value is computed and compared to the level of signiﬁcance. For Wilcoxon’s test versus the t-test this equals . The quality of a test is often expressed in terms of eﬃciency. which is usually directly related to the (asymptotic) variance of an estimator. of a few outliers or a slightly heavier tail.g.176). a low p-value indicates. If gene expression data pass a normality test. the outcomes of experiments are seen as realizations of random variables. the probability to reject a false hypothesis. that the outcome of the experiment is so unlikely that this causes a suﬃcient amount of doubt to the researcher to reject the null hypothesis.2. In case. and decisions (conclusions).68 CHAPTER 4. the Wilcoxon . under the validity of the distributional assumptions. Since there are only two such genes we can draw the reassuring conclusion that the tests give similar results.3 Overview and concluding remarks Statistical hypothesis testing consists of hypotheses. The probability of drawing correct conclusions can always be improved by increasing the sample size. Wang.05 and the absolute diﬀerence with the p-value from the t-test is larger than 0. By comparing the distribution of the statistic with the value of the statistic. 1971). which obviously should not be followed blindly. p. distributional assumptions. ESTIMATION AND INFERENCE Wilcoxon p-value is smaller than 0. Eﬃciency is directly related to power. The relative eﬃciency is the ratio of the asymptotic variances. 1999. The hypotheses pertain to the outcome of a biological experiment and are always formulated in terms of population values of parameters. However. 3. (a) Use a boxplot to construct a hypothesis about the experimental eﬀect.4. HOXA9.. EXERCISES 69 test is recommended. On NCBI there are various cDNA clones of zyxin. Gene ”MYBL2 V-myb avian myeloblastosis viral oncogene homologlike 2” has its expression values in row 1788. the p-value and your conclusion. Gene CD33. 4.4 Exercises 1. 4. (a) Test the normality of the ALL and AML expression values. . (a) Find the accession number of cDNA clone with IMAGE:3504464. (a) Test the normality of the expression values of the ALL patients. 1999). (b) Test whether the frequencies of the nucleotides are equal for each nucleic acid. it is obviously questionable whether extremely small diﬀerences in p-values produced by the t-test contribute to biologically relevant gene discrimination. Use grep to ﬁnd the index of the important gene CD33 among the list of characters golub. Zyxin. For each test below formulate the null hypothesis. (d) Is the experimental eﬀect strong? 2. can cause leukemia (Golub et al. extremely small diﬀerences should not be over-interpreted. On the other hand. (b) Test for the equality of variances. (c) Test for the equality of the means by an appropriate t-test.gnames. (b) Test for the equality of means by an appropriate t-test. Gene ”HOXA9 Homeo box A9” with expression values in row 1391.4. Because the Wilcoxon p-values are based on ranks many of these are equal for diﬀerent genes. so that it is less suitable for ordering in case of small sample size. (b) Test for the equality of means by an appropriate t-test. That is. Do you observed diﬀerence between genes? (c) Compute three measures of spread for the ALL expression values for both genes. Are there outliers? (b) Compute the mean and the median for the ALL gene expression values for both genes. . 330. 290.70 CHAPTER 4. 6. Comparing two genes. 5. Two-sample tests on gene expression values of the Golub et al. (1999) data. Scan the Golub (1999) article for genes among the ten you found and discuss their biological function brieﬂy. (1999) data. In a certain experiment the oﬀspring is observed with frequencies 930. Antigenes play an important role in the development of cancer. 90. Perform the Shapiro-Wilk normality test separately for the ALL and AML gene expression values. What percentage passed the normality test separately for the ALL and the AML gene expression values? What percentage passes both testes? 10. Report the names of the best ten. (a) Produce a boxplot for the ALL expression values and comment on the diﬀerences. (1999) data. Consider the gene expression values in row 790 and 66 of the Golub et al. Do the data conﬁrm the model? 8.1” can be predicted by the probabilities of the cDNA sequence ”BC002323. 7. Genetic Model. Order the antigenes according to their p-values from the Welch two-sample t-test with respect to gene expression values from the ALL and AML patients of the Golub et al. Gene selection.2”. (1999) data. Normality tests for gene expression values of the Golub et al. 9. Antigenes. Do you observe diﬀerence between genes? (d) Test by Shapiro-Wilk and Anderson-Darling the normality for the ALL gene expression values for both genes. ESTIMATION AND INFERENCE (c) Test whether the frequencies of ”X94991. A certain genetic model predicts that four phenotypes occur in ration 9:3:3:1. Select the genes from the golub data with smallest two-sample t-test values for which the ALL mean is greater than the AML mean. yj are values from Group 1 and 2. (b) Perform the Wilcoxon rank test and report the names of the ten genes with the smallest p-values. Programming some tests. 11. Suppose that the experiment is repeated 1000 times. Program this and illustrate it with the expression values of row 1024 of Golub et al. where xi . (b) The value of W in the two-sample Wilxoxon test equals the sum of the ranks of Group 1 minus n(n + 1)/2.4. (b) What is the probability of less than 10 rejections? (c) What is the probability of more than 5 rejections? (d) What is the probability that the number of rejections is between two and eight? 12.4. (1999) data. (c) The value of W in the two-sample Wilxoxon test equals the number of values xi > yj . (1999) data. (a) Program the two-sample t-test with equal variances and illustrate it with the expression values of row 1024 the of Golub et al. (1999) data. Suppose that the probability to reject a biological hypothesis by the results of a certain experiment is 0.05. Biological hypotheses. Program this and illustrate it with the expression values of row 1024 of Golub et al. . where n is the number of gene expression values in Group 1. respectively. (a) How many rejections do you expect. EXERCISES 71 (a) Perform the two-sample Welch t-test and report the names of the ten genes with the smallest p-values. 72 CHAPTER 4. ESTIMATION AND INFERENCE . The main focus. 73 . In this chapter the linear model will brieﬂy be explained. however. In case. is on application of the linear model for testing the hypothesis that three or more group means are equal. It is frequently applied bioinformatics. The somewhat technical concepts of “model matrix” and “contrast matrix” are explained because these are useful for several applications in the next chapter. The validity of the technique is based on the assumption that the gene expression values are normally distributed and have equal variance across groups of patients. It will be explained how the assumptions about normality and equal variances (homogeneity) can be investigated and what alternatives can be used in case either of these does not hold.Chapter 5 Linear Models We have seen that the t-test can be used to discover genes with diﬀerent means in the population with respect to two groups of patients. there are three groups of patients the question arises how genes can be selected having the largest diﬀerential expressions between group means (experimental eﬀect)? A technique making this possible is an application of the linear model and is called analysis of variance. It is of importance to investigate these assumptions because it either reassures our conﬁdence in the conclusions or it indicates that alternative tests should be used. however. Several illustrations of analyzing gene expression data will be given. The idea simply is to increase the number of weights to the number of groups k. the slope will be large. · · · . for i = 1. By choosing a few gross errors in the data it can be observed that the estimates are not robust against outliers. xi a ﬁxed number. · · · . By choosing the points nearly vertical. For a linear model to be a statistical model there must be some assumption with respect to the distribution of the error variables. εn are independent and normally distributed with zero mean. a best ﬁtting line through the data can easily be computed by least squares estimation of the intercept and slope. β an unknown weight. · · · . The model is called ”linear” because the degree of the coeﬃcient β is one. Given a gene expression Yi . Frequently. yn and x1 . A common manner to introduce the linear model is by writing Yi = β 1 + xi β 2 + ε i . we obtain the weights β1 . as we shall see.1 Deﬁnition of linear models Yi = xi β + ε i . The systematical part of the model xi β equals the mean of the gene expression Yi . a basic form of the linear model is where Yi is an observable variable. and εi the error of the model. n. so that. By choosing the points more or less on a horizontal line. Example 1. βk and the corresponding design values xi1 . A nice application to explore this is by the function put. εi a unobservable error variable. xn . according to N (0. The xi value is part of the predictor. · · · . The ﬁxed number xi follows from a statistical “design”. so that the model part represents a straight line with intercept β1 and slope β2 . the slope will be near zero. · · · . LINEAR MODELS 5. In order to handle gene expression data for three or more groups of patients we need to extend the model. Then the mean of Yi equals xi β and its variance σ 2 . xik . · · · .74 CHAPTER 5. Given data points y1 . Yi the criterion. The systematic part of the model consists of a weighted sum of these design values: . It allows points to be added and deleted to a plot which interactively computes estimates for the slope and the intercept given the data. · · · . for i = 1. n.points. it is assumed that the error variables ε1 . σ 2 ).demo() from the TeachingDemos package. that is. 9. we construct a factor indicating to which group each expression value belongs.3. and 11. DEFINITION OF LINEAR MODELS 75 xi1 β1 + · · · + xik βk .8 of Group 2.7.gl(3.9. The design values xij for Patient i in Group j are collected in the so-called ”design” matrix denoted by X. In particular. the ﬁrst four belong to Group 1.1.12 of Group 3.c(2.13. 8.1. In particular. Suppose we have the following artiﬁcial gene expressing values 2. the second four to Group 2. > a <. We may assign these to a vector y.4) > a [1] 1 1 1 1 2 2 2 2 3 3 3 3 Levels: 1 2 3 The design matrix X is also called “model matrix”. We conveniently use the function gl to deﬁne the corresponding factor. 11. This will be illustrated by an example. as follows. Example 2. By this choice it becomes possible to use linear model estimation for testing hypotheses about group means.1) a1 a2 a3 1 1 0 0 2 1 0 0 3 1 0 0 4 1 0 0 5 0 1 0 6 0 1 0 7 0 1 0 8 0 1 0 .2.5. 8. > y <.3. the design value xij is chosen to be equal to 1 if Patient i belongs to Group j and zero if (s)he does not. By adding measurement error to this systematic part we obtain the linear model k Yi = j=1 xij βj + εi .7.matrix(y ~ a . It is illuminating to print it to the screen.12.2. and the third four to Group 3.12) Next.12.8.1. > model. of Group 1.13. and Yij is the expression of Person i in Group j. estimation of the linear model comes down to estimation of group means for which there are one-sample t-type of tests available (see e. µ2 . 2003). > summary(lm(y ~ a .98e-10 *** The output in the ﬁrst column gives the estimated mean per group.0000 0.4082 19. 1 See also Chapter 11 of the manual ”An Introduction to R”. the third the t-value (the estimate divided by the standard error).394 2. the weights (β1 . To illustrate this we employ the estimation function lm and ask for a summary.09e-08 *** a3 12.0000 0. where εij is distributed as N (0.4082 4. Error t value Pr(>|t|) a1 2.000849 *** a2 8.4082 29. β2 . σ 2 ).g. . and the εij the error of Person i in Group j. From the p-values the conclusion follows to reject the null hypotheses H0 : µj = 0 for Group index j running from 1 to 3. Rao & Toutenburg. µ3 ). µj the mean of Group j. Using the above design matrix.899 0. 1995. β3 ) of the model specialize to the population means (µ1 . and for the ﬁrst member of Group 3 it is Y9 = µ3 + ε9 . Recall that population means are generally estimated by sample means. Similarly. The second gives the standard error of each mean.1 In this situation. in the current setting. The model for the ﬁrst gene expression value of Group 1 is Y1 = µ1 + ε1 .1)) Coefficients: Estimate Std.0000 0. for the ﬁrst member of Group 2 it is Y5 = µ2 + ε5 . for the second expression value of Group 1 it is Y2 = µ1 + ε2 . and the last the corresponding p-values. LINEAR MODELS The notation y~a-1 represents a model equation.76 9 10 11 12 0 0 0 0 0 0 0 0 1 1 1 1 CHAPTER 5. the model for the gene expression values from diﬀerent groups can be written as Yij = µj + εij . where -1 means to skip the intercept or general constant.596 1. The error is assumed to be normally distributed with zero mean and variance equal for diﬀerent persons. Samuels & Witmer. let three groups of patients be available with measurements in the form of gene expression values.2. In statistical language such groups are called levels of a factor. this is made possible by a technique called analysis of variance (ANOVA). · · · . The sum of squares within (SSW ) is the sum of the squared deviation of the measurements to their group mean. To set the scene. y23 . and y 3 = n n yi3 . In bioinformatics the linear model is applied to many sets of gene expressions. yn1 those of Group 2 by y12 . y22 . . where n is the number of expression values in each group. The sum of squares between (SSB) is the sum of squares of the deviances of the group mean with respect to the total mean. where g is the number of groups. The null-hypothesis to be tested is H0 : µ1 = µ2 = µ3 . yn3 . · · · . that is g n SSW = j=1 i=1 (yij − y j )2 . y21 . i=1 1 y2 = n n i=1 1 yi2 .5. N i=1 i=1 i=1 For the deﬁnition of the overall test on the equality of means there are two sums of squares of importance. so that it is of great importance to have an overall test for the equality of means. ONE-WAY ANALYSIS OF VARIANCE 77 The above illustrates that the linear model is useful for testing hypotheses about group means. 5. i=1 The total number of measurements N = 3n. yn2 and those of Group 3 by y13 . Let the data for Group 1 be represented by y11 .2 One-way analysis of variance A frequent problem is that of testing the null hypothesis that three or more population means are equal. so that the overall mean y is equal to n n n 1 y= yi1 + yi2 + yi3 . that is g n g SSB = j=1 i=1 (y j − y)2 = n j=1 (y j − y)2 . By comparing two types of variances. The three sample means per patient group can be expressed by 1 y1 = n n yi1 . · · · . ssb <. then this f -value follows the Fg−1. mean)) g <. 1973.12) > a <.0 for (j in 1:g) {ssb <. and y 3 = 12.mean(y))^2} SSB <. Example 1. The number of expression values per group n = 4. These group means are now collected in the vector gm. The grand mean y can be computed by mean(y)=7.245).c(2. where g − 1 and N − g are the degrees of freedom (Rao. 12. SSW/(N − g) If the data are normally distributed. 2. and.2.numeric(tapply(y.as.0 > for (j in 1:g) {SSW <. If P (Fg−1.12. 7. to construct the corresponding factor.(SSB/(g-1))/(SSW/(N-g)) . and the number of groups g = 3.3. 9.7.6667.n*ssb This results in SSB = 202. then H0 : µ1 = µ2 = µ3 is not rejected. those of Group 2 are 8.13. a.N −g > f ) ≥ α.4. y 2 = 8. p. Recall that the data of Group 1 are 2. and to compute the group means one may use the following. To load the data. 13.gl(3. > y <. mean)) > gm [1] 2 8 12 Thus we ﬁnd that y 1 = 2.78 Now the f -value is deﬁned by f= CHAPTER 5. n <. In a similar manner the sums of squares within (SSW ) and the f -value can be computed. 8.8. LINEAR MODELS SSB/(g − 1) . An elementary manner to compute the sums of squares between SSB is by gm <.numeric(tapply(y. N <-12.1.N −g distribution. a.3.4) > gm <.9. and of Group 3 are 11. 12. The idea behind the test is that. so that the observed f -value will be small and H0 is accepted. the total number of data values N = 12. 3.333333. otherwise it is. the value for SSB will tend to be small.SSW + sum((y[a==j]-gm[j])^2)} > f <. as follows. under the null-hypothesis of equal group means. 1. 11. > SSW <. 8.as.ssb + (gm[j]. Let’s continue with the data from the previous example. 2.159 · 10−7 .4082 4. the conclusion is to reject the null hypothesis of equal means.000 0. the sums of squares within (6.0000 0. 9) = 1. Such corresponds to the following contrast matrix   1 1 1 C =  0 −1 0  . not clear which of the means diﬀer.9 < 152) = 1 − pf(152.667 This gives the degrees of freedom g − 1 = 2 and N − g = 9.9 > 152) = 1 − P (F2.0). Since this is smaller than the signiﬁcance level 0. and the diﬀerence between Group 3 and Group 1. It is. The built-in-function anova can be used to extract the so-called analysis of variance table from an lm object.667 101. ONE-WAY ANALYSIS OF VARIANCE 79 This results in SSW = 6 and an observed f -value equal to 152. as follows. 0 0 −1 This contrast matrix is by default implemented by the model speciﬁcation y~a.899 0. the f -value 152 and the overall p-value 1. the sums of squares between (202.159e-07 *** Residuals 9 6. > summary(lm(y ~ a)) Coefficients: Estimate Std. Error t value Pr(>|t|) (Intercept) 2. Example 2.333 152 1. By the previous analysis of variance it is concluded that there are diﬀerences in population means.667). the overall p-value is P (F2.2.05.5.000849 *** .159156 · 10−7 . > anova(lm(y ~ a)) Analysis of Variance Table Response: x Df Sum Sq Mean Sq F value Pr(>F) fact 2 202. Hence. however. A way to clarify this is by estimating the mean of Group 1 (Level 1) and then computing the diﬀerence between Group 2 and Group 1. 13 0.1. and H0 : µ3 − µ2 = 0.9649 F-statistic: 152 on 2 and 9 DF. the null-hypotheses are H0 : µ1 = 0. By t-tests the null-hypothesis is tested that the mean of Group 1 is zero.35 1.2) [1] 1. all null-hypotheses are rejected. That is.5774 CHAPTER 5.9.61 2.75 1. The last line of the output gives the f -value.5774 0. Example 3.80 2. and the corresponding overall p-value.4) > anova(lm(y ~ a))$Pr[1] [1] 0. p-value: 1.gl(3.392 2. The latter equals that of ANOVA.08 1.00 > a <.321 3. Adjusted R-squared: 0.5) > round(x.27 2. H0 : µ2 − µ1 = 0.0000 0.159e-07 The estimated intercept is the mean of Group 1 (Level 1). LINEAR MODELS 10.0.80 factLevel 2 factLevel 3 6.8165 on 9 degrees of freedom Multiple R-Squared: 0.05. which is consistent with the data generation process. The p-value implies the conclusion not to reject the null-hypotheseis of equal means.50 2. the diﬀerence in means between Group 2 and Group 1 is zero and the diﬀerence in means between Group 3 and Group 1 is zero.0000 10.rnorm(12.9 and standard deviation 0.6154917 Note that by the $Pr[1] operator extracts the p-value from the list generated by the anova function.5 corresponding to three groups of patients that do not possess any type of diﬀerences between groups. The factLevel 2 is the diﬀerence in means between Group 2 (Level 2) and Group 1 and factLevel 3 is the diﬀerence in means between Group 3 and Group 1.9712. the degrees of freedom. > y <. Before we analyze real gene expression data it seems well to give an example where the means do not diﬀer.40 2.60e-06 *** 17. .22e-08 *** Residual standard error: 0.82 1.71 2. Let’s sample data from the normal distribution with mean 1. Since the p-values that correspond to the t-values are smaller than the signiﬁcance level 0. 873 < 2e-16 *** (Intercept) .5 B1 B2 6.2: Plot of Ets2 expression values for three patient groups.0 4.5 5."B2". expression levels from B-cell ALL patients in stage B1. The null hypothesis is tested that the expression means in each stage are equal or in other words that there are no experimental eﬀects.08506 53.] summary(lm(y ~ ALLB123$BT)) Estimate Std. To illustrate analysis of variance by real data we shall use the ALL data from the ALL package.2.4 6. B-cell ALL: 1866_g_at. It is brieﬂy indicated how the data are loaded.1.0 3. Example 4.0 B3 B1 6. and B3 are selected with row name 1866_g_at.5.6 6.2 B2 B3 Figure 5.1: Plot of SKI-like oncogene expressions for three patient groups.5 4.exprs(ALLB123)["1866_g_at".1 it can be observed that the expression levels diﬀer between the disease stages. Speciﬁcally.8 7."B3")] y <.2 6. Error t value Pr(>|t|) 4. > > > > library(ALL). ONE-WAY ANALYSIS OF VARIANCE 81 5.ALL[.58222 0. which refers to an SKI-like oncogene related to oncoproteins. Figure 5. From the plot of the data in Figure 5.0 7. B2.data(ALL) ALLB123 <.ALL$BT %in% c("B1". see Section 1. More speciﬁcally.10513 0."B2".11494 CHAPTER 5. To illustrate a case where the means do not diﬀer we selected the expression values for probe 1242_at of the Bcell ALL patients in stage B1.636 the H0 : µ2 − µ1 = 0 is not rejected."B3")] y <.exprs(ALLB123)["1242_at".07665 -0. B-cell ALL: 1242_at.610 0.636 ALLB123$BTB3 -0. the population means of Group B1.00e-08 *** Residual standard error: 0. LINEAR MODELS -4. The data are extracted from the ALL object and collected in the vector y.207 · 10−7 of the f -test the conclusion follows to reject the hypothesis of equal means. B2.207e-07 From the overall p-value 1. however. Adjusted R-squared: 0. p-value: 1.82 ALLB123$BTB2 -0.07011 0.156 8. and B3 from the ALL data.3287 F-statistic: 19.483 <2e-16 *** ALLB123$BTB2 0. Example 5.04675 0. p-value: 0.475 0. Error t value Pr(>|t|) (Intercept) 6.ALL[. the conclusion is not to reject the null hypothesis of equal means. B2.3707 on 75 degrees of freedom Multiple R-squared: 0.55083 0. .2.2473 on 75 degrees of freedom Multiple R-squared: 0.85 on 2 and 75 DF. > > > > library(ALL). From the t-tests we conclude that the mean of B1 diﬀers from zero and the diﬀerences between B2 and B1 as well as between B3 and B2 are unequal to zero.006898 F-statistic: 0.3461. Adjusted R-squared: -0.52e-05 *** -6. data(ALL) ALLB123 <. From the plot of the data in Figure 5.4823 From the overall p-value 0.72193 0. and B3 do diﬀer.] summary(lm(y ~ ALLB123$BT)) Estimate Std.43689 ALLB123$BTB3 -0.03331 0. the null-hypotheses H0 : µ1 = 0 is rejected.01925. This probe corresponds to the Ets2 repressor factor which plays a role in telomerase regulation in human cancer cells.7362 on 2 and 75 DF.544 Residual standard error: 0. That is.ALL$BT %in% c("B1".05673 115. but from the p-value 0.4823. The corresponding factor is given by ALLB123$BT. it can be observed that the expression values hardly diﬀer between disease stages.281 2.

This is indeed possible."B4") & ALL$mol.ALL[. > pano <.544 the H0 : µ3 − µ2 = 0 is not rejected either.5."B2". the question may arise whether the model for means of groups can be extended from one factor to more factors. data(ALL) ALLBm <. TWO-WAY ANALYSIS OF VARIANCE and from p-value 0. If the means of the i groups diﬀer. then there is a main eﬀect of the ﬁrst factor which is expressed in a p-value smaller than 0. It case of the ALL data from Chiaretty et al.3.05) [1] 2526 Thus the hypothesis of equal means is rejected for 2526 out of a total of 12625 genes (probes)."B3".05. (αβ)ij the interaction eﬀect and εijk the error which is distributed according to N (0. Two-way analysis of variance will brieﬂy be illustrated. 83 Example 6. library("ALL").which(ALL$BT %in% c("B".biol %in% c("BCR/ . Similarly. We shall perform the analysis on the expression values of NEDD4 binding protein 1 with probe id 32069_at. (2004) we may aggregate the B cell patients into two groups: B.3 Two-way analysis of variance Having some experience with one way analysis of variance. B1 and B2 in the ﬁrst and B3 and B4 in the second. there is a main eﬀect of the second factor. βj the mean of Group j indicated by the second factor.function(x) anova(lm(x~ALLB123$BT))$Pr[1]) > sum(pano<0. σ 2 ). This can be computed as follows. For the second group we select from the molecular biology of the patients assigned to BCR/ABL and NEG. The model would then be equal to Yijk = αi + βj + (αβ)ij + εijk . where αi is the mean of Group i indicated by the ﬁrst factor."B1".apply(exprs(ALLB123). expressed in a p-value smaller than 0. in case the means of the j groups diﬀer. An interesting question is of course for how many genes of the ALL data the hypothesis of equal means is rejected by the overall ANOVA p-value? Such can be answered by collecting the p-values in a vector. 5.1. A two-way approach.05. Example 5.

0341 * Residual standard error: 0.0006433 *** facB:facmolb 1 1. function(x) anova(lm(x ~ facB * facmolb))$Pr[ .316 on 3 and 75 DF."B34 > anova(lm(exprs(ALLBm)["32069_at".1659 1. One may also ask for a summary of the individual eﬀects.4e-05 *** facBB34:facmolbNEG 0.7649 0.6020 0. ] Df Sum Sq Mean Sq F value Pr(>F) facB 1 1. 1.factor(ceiling(as.2535 First the patients are selected with B-cell ALL and assigned molecular biology of the cancer to BCR/ABL or NEG.labels=c("B012".5231 0. From the p-values in the analysis of variance table it can be concluded that there two main eﬀects as well as an interaction eﬀect.159 0.103 0. Error t value Pr(>|t|) (Intercept) 6. In this case we may compute the number of probes with signiﬁcant main as well as interaction eﬀects.1073 63.0352127 * facmolb 1 3. > pval <.] ~ facB * facmolb)) Analysis of Variance Table Response: exprs(ALLBm)["32069_at".1809 4.2162 3.0027 ** facmolbNEG -0.2162 12.6592 0. > summary(lm(exprs(ALLBm)["32069_at".0340869 * Residuals 75 19.1659 4.integer(ALLBm$BT)/3).1809 1.5999 0. LINEAR MODELS facmolb <.levels=1:2.2324 2. p-value: 0.] ~ facB * facmolb)) Call: lm(formula = exprs(ALLBm)["32069_at".5016 0.biol) facB <.84 CHAPTER 5.2264.0002285 In bioinformatics the question often arises how many probes there are with have signiﬁcant eﬀects.1686 -3.1954 F-statistic: 7. Next the factors are constructed to group the patients.128 9.6891 0.1458 -4.0094 0.factor(ALLBm$mol.5034 on 75 degrees of freedom Multiple R-squared: 0. ] ~ facB * facmolb) Coefficients: Estimate Std. Adjusted R-squared: 0.apply(exprs(ALLBm).026 < 2e-16 *** facBB34 -0. This matrix is transposed so that the columns correspond to the p-values and the rows to the probes. The normality of the residuals from the estimated linear model on the B-cell ALL data from 1866_g_at. The latter is generally known as the homoscedasticity assumption.0005989 .05 & pvalt$maineffectmolbiol < 0. The homoscedasticity assumption can be tested as a hypothesis by the Breusch and Pagan (1979) test on the residuals. From Figure 5."interaction") > sum(pvalt$maineffectB < 0.4 Checking assumptions When the linear model is applied for analysis of variance there are in fact two assumptions made.library(ALL) ALLB123 <.ALL[.] shapiro.exprs(ALLB123)["1866_g_at". CHECKING ASSUMPTIONS 85 > pvalt <.data.05 & pvalt$interaction < [1] 47 The three p-values per probe are collected in a matrix.1 it can be observed that there are outliers being far apart from the bulk of the other expression values. First. 5.ALL$BT %in% c("B1". > > > > data(ALL. second."maineffectmolbiol".5.c("maineffectB"."B2". the errors are assumed to be independent and normally distributed.package="ALL"). Using the logical AND (&) operator and summing the TRUE values yield 47 probes with signiﬁcant main and interaction eﬀects. p-value = 0. and. Testing normality of the residuals.4.9346. The normality assumption can be tested as a null hypothesis by applying the Shapiro-Wilk test on the residuals. the error variances are assumed to be equal for each level (patient group). can be tested as follows. This latter test may very well be seen as a generalization of the F -test for equal variances.frame(t(pval)) > colnames(pvalt) <."B3")] y <.test(residuals(lm(y ~ ALLB123$BT))) Shapiro-Wilk normality test data: residuals(lm(y ~ ALLB123$BT)) W = 0. Example 1. This raises the question whether the normality assumption holds. 5.library(ALL) . library(lmtest) ALLB123 <.1 it can be observed that the spread of the expression values around their mean diﬀers between groups of patients. > data(ALL.] bptest(lm(y ~ ALLB123$BT). the conclusion follows to reject the null hypothesis of equal variances (homoscedasticity). the null hypothesis H0 : µ1 = µ2 = µ3 of equal means can be tested without assuming equal variances by a test proposed by Welch (1951). Example 1.exprs(ALLB123)["1866_g_at". In Example 2 of the previous section the hypothesis of equal variances was rejected. From Figure 5.86 CHAPTER 5.studentize = FALSE) Breusch-Pagan test data: lm(y ~ ALLB123$BT) BP = 8.test.ALL$BT %in% c("B1". we are in a situation quite similar to that of ttesting with unequal variances. In order to test the homoscedasticity assumption we use the function bptest from the lmtest package. LINEAR MODELS From the p-value 0. p-value = 0. > > > > library(ALL)."B3")] y <. an alternative testing procedure is called for."B2".ALL[.7311.package="ALL"). To apply analysis of variance without assuming equal variances (homoscedasticity) one may use the function oneway.0005989. as follows. That is. In case only homoscedasticity is violated. the conclusion is to reject the null-hypothesis of normally distributed residuals.5 Robust tests In case departures from normality or homoscedasticity are large enough to cause concern with respect to the actual signiﬁcance level or to the power of the test.01271. Example 2. data(ALL).01271 From the p-value 0. df = 2. Testing homoscedasticity of the residuals.

does not estimate the size of experimental eﬀects. We use the function kruskal. > > > > data(ALL.test(y ~ ALLB123$BT) 87 One-way analysis of means (not assuming equal variances) data: y and ALLB123$BT F = 14. the Kruskal-Wallis rank sum test is recommended.000. In case normality is violated a rank type of test is more appropriate. ROBUST TESTS > ALLB123 <."B3")] > y <.1573. num df = 2.5. p-value = 2. Because it is based on ranking the data.998."B2".ALL$BT %in% c("B1". the null-hypothesis of equal distributions of expression values between patient groups is rejected. df = 2. denom df = 36. In Example 1 of the previous section we rejected the hypothesis of normally distributed residuals. .ALL[."B3")] y <.ALL$BT %in% c("B1". it is highly robust against non-normality.package="ALL").test(y ~ ALLB123$BT) Kruskal-Wallis rank sum test data: y by ALLB123$BT Kruskal-Wallis chi-squared = 30. p-value = 2.exprs(ALLB123)["1866_g_at".] > oneway. Example 2.6666. By the apply functionality the p-values can easily be computed for all 12625 gene expression values of the ALL data."B2". however. This test can very well be seen as a generalization of the Wilcoxon test for testing the equality of two distributions.717e-05 From the p-value 2.] kruskal.192 · 10−7 .test to perform a non-parametric test. to test the null-hypothesis of equal distributions of groups of gene expression values. In particular.717 · 10−5 .ALL[.5. the conclusion follows to reject the hypothesis of equal means.192e-07 From the p-value 2. it.exprs(ALLB123)["1866_g_at".library(ALL) ALLB123 <.

5. B3. B1. In the next chapter it will brieﬂy be indicated how to combine two factors into a single analysis of variance. (d) Do the same for the homoscedasticity assumption. For instance. Here. (b) How many patients are in each group. LINEAR MODELS 5. More examples are given in the next chapter where several functionalities of Bioconductor will be used for the analysis of microarray data. (c) Test the normality of the residuals from the linear model used for analysis of variance for all gene expression values. B4 from the ALL data.88 CHAPTER 5.7 Exercises (a) Construct a data frame containing the expression values for the B-cell ALL patients in stage B. 2. The p-values from overall tests of equality of means or distributions are important tools to order genes according to their experimental eﬀect with respect to diﬀerent patient groups. false positives are signiﬁcant p-values for equal populations means and false negatives are non-signiﬁcant p-values for unequal populations means. B2. Collect the p-values in a vector. Further analysis of gene expressions of B-cell ALL patients.6 Overview and concluding remarks By applying the above normality and homogeneity tests to complete sets of gene expression values it can quickly be seen to what extent the assumptions for the classical analysis of variance test are violated. Based on these it can be decided to add rank type of testing in order to reduce the amount of false positives and false negatives. Analysis of gene expressions of B-cell ALL patients. The interested reader is referred to Faraway (2004) and Venables & Ripley (2002) for more information on using linear models in R and for a general treatment of linear models to Rao & Toutenburg (1995). one may want to combine B-cell stage with age groups of persons. Continue with the previous data frame containing the expression values for the . (e) How many gene expressions are normally distributed and how many homoscedastic? For how many do both hold? 1.

B2. (d) Use featureNames() to report the aﬀymetrix id’s of the genes with smaller p-values than 0. then the conclusion is that there an experimental eﬀect (a positive). (c) Use the function intersect to ﬁnd identiﬁers in both sets. (b) Do the same for the p-values from the Kruskal-Wallis test. B3. How many false positives do you expect and how many did you observe? .05? (d) If the p-value is smaller than the signiﬁcance level. (a) Construct a data matrix with 10000 rows and 9 columns with data from the normal distribution with mean zero and variance equal to one. A simulation study on gene expression values. B2. B4 from the ALL data. B3. 89 (b) Use featureNames() to report the aﬀymetrix id’s of the genes with smaller p-values than 0. That is. (c) How many p-values are smaller than the signiﬁcance level α = 0. EXERCISES B-cell ALL patients in stage B. (c) Collect the overall p-values from the Kruskal-Walles test in a vector.000001.7. (b) Construct a factor for three groups each with three values. (a) Print the p-values and the corresponding (aﬃmetrix) gene identiﬁers of the ten best from ANOVA.001 from both ANOVA and Krusal-Wallis? How many only from one type of test? Hint: Collect TRUE/FALSES in logical vectors and use table. Finding the ten best best genes among gene expressions of B-cell ALL patients. B4 from the ALL data.5. 4. 3. B1. (e) Brieﬂy comment on the diﬀerences you observe. Continue with the previous data frame containing the expression values for the B-cell ALL patients in stage B. (a) Collect the overall p-values from ANOVA in a vector. how many genes have p-values smaller than 0. Such a matrix simulates gene expressions without diﬀerences between groups (sometimes called negatives).000001. B1.

Use ANOVA and kruskal-Wallis to ﬁnd the number of signiﬁcant genes (true positives). Assume again that there three groups each with three data values. report the number of true positives and false negatives. one and two and variance equal to one. LINEAR MODELS (e) Construct a matrix with 10000 rows and 9 columns with normally distributed data with mean zero.90 CHAPTER 5. This data matrix simulates gene expressions with diﬀerences between groups (sometimes called positives). .

complementary molecules are attached and labeled on a solid surface in order for a specialized scanner measure the intensity of target molecules. The technique makes it possible to give an initial answer to many important genetic type of questions. The intensity of the perfect match (PM) intends to measure the amount of transcripts from the gene.1 Probe data The microarray technique takes advantage of hybridization properties of nucleic acids.Chapter 6 Micro Array Analysis The analysis of gene expression values is of key importance in bioinformatics. where we will work with. 1 91 . as well as how to summarize results in html output. 1 6. ﬁlter genes. which are processed to so-called CEL ﬁles. will be to concentrate on the programming aspects using the commandline. however. to load public available gene expression data. to use gene ontology identiﬁers. Our approach. It may be convenient to explore the possibilities of the limmaGUI. The package affy has facilities to read data from a vector specifying several CEL ﬁles produced by the Aﬀymetrix scanner. In this chapter you learn how to preprocess probe data. Per gene there are about twenty such measures obtained for each probe (gene). The raw data from the Aﬀymetrix scanner is stored in so-called DAT ﬁles. to program various visualizations. to give a rough idea. The intensity of the mismatch (MM) is related to non-speciﬁc binding and is often seen as a background type of noise. That is. Per probe these measures come in pairs.

B) [1] "cdfName" [4] "assayData" [7] "experimentData" "nrow" "phenoData" "annotation" "ncol" "featureData" ".B can be obtained by the dim function. > dim(exprs(MLL. The PM and MM values are collected by the functions pm and mm.B) [1] "hgu133b" To print the ﬁrst 10 names of the probes use > probeNames(MLL. > annotation(MLL. which extracts the probe intensities from the MLL.B from the ALLMLL package. We will start with a built-in data set called MLL. The raw probe intensities are available from exprs(MLL. The number of rows and columns of the expression values of MLL. To load it and to retrieve basic information use > library(affy) > data(MLL. > slotNames(MLL.B)) [1] 506944 20 The annotation can be extracted as follows. .B It is very useful to print the structure of the object str(MLL.B.B)[1:10] [1] "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" [6] "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" "200000_s_at" Note that the probe names are the same as those obtained by geneNames.B) and its slot names.B). MICRO ARRAY ANALYSIS Example 1.B object. package = "ALLMLL") > MLL. To print the PM values of the ﬁrst four out of the sixteen rows of the probe with identiﬁer 200000_s_at we may use the following.92 CHAPTER 6.B).__classVersion__" Additional information become available from str(MLL.

6 0.2 0. + ylab="PM Probe intensity") From the resulting plot in Figure 6.B. > matplot(pm(MLL.0 0.2 6 8 10 log intensity 12 14 Figure 6.5 200000_s_at2 838.B data. plot.3 275.3 395.6.8 By function matplot a quick view on the variability of the data within and between probes can be obtained.1: Mat plot of intensity values for a probe of MLL.5 321.5 341.2: Density of MLL. Figure 6. The script to program such plots 2000 1500 PM Probe intensity density 2 4 6 Probe No.4 0.2 it can be seen that these are quite skew to the right.3 200000_s_at4 425.method= "smoothScatter") > image(MLL. PROBE DATA 93 > pm(MLL.type="l".8 253.".8 1."200000_s_at")."200000_s_at")[1:4.1:3] JD-ALD009-v5-U133B.B.CEL 200000_s_at1 661.8 409. From the density plot of the log of the intensity data in Figure 6. 8 10 1000 500 0.1 it can be observed that the variability is substantial.B.0 1. is quite brief.3 200000_s_at3 865. Density plots of the log of the probe values can be obtained by hist(MLL.CEL JD-ALD051-v5-U133B. xlab="Probe No.B. > MAplot(MLL.5 196.1.B) .B).5 312.CEL JD-ALD052-v5-U133B.pairs=TRUE.

and robust quantiles. qspline. so that deﬁnitive recommendations are not mandatory. To obtain the available background and pm correction methods use the following. MICRO ARRAY ANALYSIS 6. Loess is a nonlinear method based on local regression of MA plots. Preprocessing consists of three major steps: Background correction.methods(MLL. and is based on conditional expectation and the normality assumption of probes values.2 Preprocessing methods From various visualization methods it is clear that preprocessing of probe intensities is necessary for making biologically relevant conclusions. Quantile normalization is an inverse transformation of the empirical distribution with respect to an averaged sample quantile in order to impose one and the same distribution to each array. The method qspline uses quantiles from each array and a target array to ﬁt a system of cubic splines.94 CHAPTER 6. > bgcorrect. .methods [1] "mas" "none" "rma" "rma2" The mas background is part of the MAS Aﬀymetrix software and is based on the 2% lowest probe values. Bioconductor gives facilities for various preprocessing methods. and summarization. but could also be the name of a particular group.methods [1] "mas" "pmonly" "subtractmm" The following normalization methods are available: > normalize.B) [1] "constant" "contrasts" [5] "qspline" "quantiles" "invariantset" "loess" "quantiles. RMA uses only the PM values. quantiles. Here we will only sketch what the main methods are and how these can be implemented. It should be noted that the topic of optimal preprocessing currently is a ﬁeld of intense research (probably for the coming years). normalization.robust" Constant is a scaling method equivalent to linear regression on a reference array although without intercept term. The methods of contrasts is based on loess regression. There are also a number of correction methods available for the PM values: > pmcorrect. The target should be the mean (geometric) or median of each probe. neglects the MM values totally. More general are the non-linear normalization methods such as loess.

testing for a gene to have mean expression value . It combines convolution background correction. summary.B.method="pmonly".2. PREPROCESSING METHODS 95 The ﬁnal step of preprocessing is to aggregate multiple probe intensities into a gene expression value. for instance. Example 1. Another frequently applied preprocessing method is RMA. quantile normalization.method="rma". however.summary. eset <.bgcorrect. Before a box-and-whiskers plot can be constructed the expression values need to be extracted from the object eset3. package = "ALLMLL") > eset3 <.stat.method="constant".rma(MLL. > library(affy) > data(MLL. wise to use methods robust against outliers together with non-linear normalization methods.B. It seems.frame(exprs(eset3))) The three stages of preprocessing by rma are part of the output. When the patient median is zero.expresso(MLL.pmcorrect. After the foregoing it is often desirable to further preprocess the data in order to remove patient speciﬁc means or medians. and summarization based on multi-array model ﬁt in a robust manner by a socalled median polish algorithm.B) Background correcting Normalizing Calculating Expression > boxplot(data. The three pre-processing steps can be employed one after the other by the function expresso. normalize. There is no single best method for all preprocessing problems. The available methods are: > express. we may use the following.method="avgdiff") Example 2.6.methods [1] "avgdiff" "liwong" "mas" "medianpolish" "playerout" The ﬁrst is the simplest as it is based on averaging. To combine the background correction RMA with constant normalization and to use average diﬀerences for the computation of gene expression values.

FUN="/") "1005_at" . which is useful for further analysis. In case the gene expression values over the patients are non-normally distributed one may want to subtract the median and divide by the MAD. mad) meds <. 2. median) dat <. For instance.__classVersion__" > row.1) and further processing steps are illustrated. package = "ALL") > slotNames(ALL) [1] "assayData" "phenoData" "featureData" [4] "experimentData" "annotation" ".ALL$mol == "ALL1/AF4"] mads <.sweep(exprs(ALL1). ALL1pp <. (2004). 2. One can also ask for table(ALL$BT) to obtain an overview of the numbers of patients which are in certain phases of a disease. Here the data set is brieﬂy introduced (see also Section 1. mads. the ALL$mol variable has TRUE/FALSE values for each of the 128 patients depending on whether a reciprocal translocation occurred between the long arms of Chromosome 9 and 22. 2.apply(exprs(ALL1). next. 12625 gene expression values are available from microarrays of 128 diﬀerent persons suﬀering from acute lymphoblastic leukemia (ALL).apply(exprs(ALL1).ALL1 <. See also the general help ?ALL for further information on the data or the article by Chiaretti et al. > data(ALL. to divide each column entry by the MAD.pData(ALL) phenotypical information from the patients is stored in a data frame.ALL[.96 CHAPTER 6. In the sequel we shall frequently work with the ALL data from the ALL package of Bioconductor. An eﬃcient manner to do so is to use an apply function to compute the column mad and median.names(exprs(ALL))[1:10] [1] "1000_at" "1001_at" "1002_f_at" "1003_s_at" "1004_at" [7] "1006_at" "1007_s_at" "1008_f_at" "1009_at" By feno <. Example 3. A number of interesting phenotypical co-variates are available. and sweep to subtract the median from each column entry and.sweep(dat. The raw data have been jointly normalized by RMA and are available in the form of an exprSet object. 2. MICRO ARRAY ANALYSIS diﬀerent from zero becomes meaningful. This is casually related to chronic and acute leukemia. meds) exprs(ALL1pp) <. The examples stress the importance of careful thinking. By comparing the box plots in Figure 6.6.4 the eﬀect of preprocessing can be observed. GENE FILTERING 97 By this script the patients are selected with assigned molecular biology equal to ALL1/AF4.3 Gene ﬁltering A few important methods to ﬁlter genes are illustrated here.4: Boxplot of the ALL1/AF4 patients after median subtraction and MAD division. 14 12 10 8 6 4 2 X04006 X16004 X24005 X28028 X31007 −1 X04006 0 1 2 3 4 X16004 X24005 X28028 X31007 Figure 6. . Then the ﬁrst sweep function subtracts the medians from the expression values and second divides these by the corresponding MAD. of the Figure 6.3 and 6. Note that by box plotting a data frame a fast overview of the distributions of columns in a data frame is obtained. 6. The median and the MAD are computed per column by the speciﬁcation 2 (column index) in the apply function. Then ALL1 is copied in order to overwrite the expression values in a later stage.3: Boxplot ALL1/AF4 patients.3. The medians of the preprocessed data are equal to zero and the variation is smaller due to the division by their MAD. It is wise to keep in mind that there are statistical as well as and biological criteria for ﬁltering genes and that a combination of these often gives the most satisfactory results. function(x){sd(x)/abs(mean(x))}) Now using sum(cvval<0.f5.test(x)$p.975. Filtering by the coeﬃcient of variation. cv < 0.filterfun(f1.9)) ff <. the ﬁfth if the coeﬃcient of variation is smaller than 0. Combining several ﬁlters.1. the second if 25% of the gene expression values is larger than 6.2. Example 2.f4. If cv = 1. the fourth if it passes the Shapiro-Wilk normality test. then the mean is ﬁve times larger than the standard deviation.function(x) (sqrt(10)* abs(mean(x))/sd(x) > qt(0.25. The script in this example is useful when several functions are to be applied to a single data set. Of course it is possible to program ﬁlters completely on your own.f2. MICRO ARRAY ANALYSIS Example 1. It is often desired to combine several ﬁlters. ff) After running this script and using sum(selected) one obtains 317 genes that pass the combined ﬁlter.98 CHAPTER 6.2.function(x) (median(2^x) > 300) f4 <.1.pOverA(. which is deﬁned as the standard deviation divided by the absolute value of the mean: cv = σ/|µ|. log2(100)) f3 <.5) f2 <. however. A manner to ﬁlter genes is by the coeﬃcient of variation.function(x) (sd(x)/abs(mean(x))<0.2) yields 4751 genes with a coeﬃcient of variation smaller than 0.643856. then the standard deviation equals the mean.apply(exprs(ALL1pp). however. Let’s compute the coeﬃcient of variation per gene for the ALL1pp data of the previous section. > cvval <.function(x)(IQR(x)>0.5.function(x) (shapiro. These genes can be selected by ALL1pp[cvval<0. The ﬁlter .f3. data(ALL) selected <.2. the third if the median of the expression values taken as powers to the base two is larger than 300. The ﬁrst function returns TRUE if the interquartile range is larger than 0.f6) library("ALL"). so that both the experimental eﬀect and the measurement precision are large. library("genefilter") f1 <.value > 0. we may conveniently use the function filterfun to combine several ﬁlters.1) f6 <.].genefilter(exprs(All[.05) f5 <. so that the experimental eﬀect is small relative to the precision of measurement. If. and the sixth if the one-sample t-value is signiﬁcant.ALL$BT=="B"]). 3. sel2.library("ALL"). data(ALL) patientB <. For these genes it holds that the expression values for B-cell ALL patients as well as for T-cell ALL patients are normally distributed (in the sense of non-rejection)."B1". filterfun(f2)) selected <.value > 0. the ﬁrst ﬁlter selects genes with a certain minimal standard deviation.6. library("genefilter").ALL[selected.genefilter(exprs(ALL[.05) sel1 <. since the IQR divided by 1.patientB==TRUE]).1 is equivalent to 10|x|/s > 1/ 10.patientB==FALSE]). which is √ highly similar √ the second ﬁlter.genefilter(exprs(ALL[. as well as sel3 have the value TRUE. we create a logical factor patientB indicating patients with B-cell ALL (TRUE) and with T-cell ALL (FALSE). The latter will be applied separately for the B-cell ALL patients and for the T-cell ALL patients.sel1 & sel2 & sel3 ALLs <. s/|x| < 0. First. With respect to the third ﬁlter note that 2x > 300 is equivalent to x > 2 log(300) ≈ 8. A logical variable named selected is deﬁned which attains TRUE only if sel1. GENE FILTERING 99 functions are combined by filterfun and the function genefilter returns a logical vector indicating whether the gene passed all the ﬁlters or failed at least one of them. In order to use these ﬁlter steps properly it is well to think them through because several ﬁlters focus on similar properties.test(x ~ patientB)$p.genefilter(exprs(ALL). Filtering by t-test and normality. For this we write a function that will be used twice."B2". however.228819. so that the last two ﬁlters are highly similar. The ﬁlter deﬁned selects genes that have their p-value from the Welch two-sample t-test smaller than the signiﬁcance level 0."B4")) f1 <."B3". filterfun(f1)) sel2 <.function(x) (shapiro.test(x)$p.] This gives 1817 genes which pass the three ﬁlters. This can be combined with a normality test in the sense that only those genes are ﬁltered which pass the Shapiro-Wilk normality test. One may also want to select genes with respect to p-values of a two-sample t-test over B-cell ALL versus T-cell ALL.05) f2 <.function(x) (t.factor(ALL$BT %in% c("B".349 is a robust estimator of the standard deviation. to Furthermore. A fundamental manner to visualize how the genes are divided among ﬁlters is . Example 3. In particular.05. filterfun(f1)) sel3 <.value < 0.

library(limma) x <.c("sel1". sel1 sel2 826 1817 359 1366 920 sel3 1780 −1 X04006 0 1 2 3406 2151 3 4 X16004 X24005 X28028 X31007 Figure 6.matrix(as.ncol = 3."sel3") vc <.5 it can be seen that 1817 genes pass all three ﬁlters. This can conveniently be done by using functions from the limma package (Smyth.sel2. Figure 6. MICRO ARRAY ANALYSIS by construction of a Venn diagram. such as ANOVA.6: Boxplot of the ALL1/AF4 patients after median subtraction and MAD division.vennCounts(x. 2005). etc. 6.byrow=FALSE) colnames(x) <.5: Venn diagram of seleced ALL genes. .integer(c(sel1. include="both") vennDiagram(vc) From the resulting Venn diagram in Figure 6.sel3)). 1780 genes pass none.100 CHAPTER 6. 3406 genes pass the normality tests but not the t-test ﬁlter.4 Applications of linear models The limma package is frequently used for analyzing microarray data by linear models."sel2".

2004)2 . APPLICATIONS OF LINEAR MODELS 101 Example 1.lmFit(allB.333e-97 2773 32748_at 12. Such a speciﬁc hypothesis can be tested by using a contrast matrix.topTable(fit. Analysis of variance.45 295.B2 B 1 0 B1 -1 1 B2 0 -1 2 To obtain the appropriate number of levels we make a factor of ALLB$BT. . coef=2.eBayes(fit) > toptab <. Rather.B1 B1 .ma <. We select patients with B-cell leukemia in a beginning stage B and in more progressive stages B1 and B2.771e-97 5328 35278_at 12."B1".B1-B2."B2"))] design. library("ALL").3 1.5 1.0 4.6.ma <. because this is the diﬀerence between Stage 0 and Stage 3.digits=4) ID logFC AveExpr t P. levels=factor(allB$BT)) > cont."B1".50 326.ma) fit <.method="fdr") > print(toptab[.ma) <. which can be speciﬁed as follows.146e-96 4636 34593_g_at 12.44 12.70 306. Let’s call the mean of the B patients µ. we are interested in the hypothesis H0 : µ − µ1 and H0 : µ1 − µ2 .5.Value 12586 AFFX-hum_alu_at 13."B2") fit <.model. In the current case we are not so much interested in the hypothesis H0 : µ − µ2 .3 9. Then the linear model is ﬁtted to the data and an empirical Bayes procedure is used to adapt the gene speciﬁc variances with a global variance estimator (Smyth.makeContrasts(B-B1.431e-95 By topTable the ﬁve genes are selected with the smallest p-values adjusted for the false discovery rate. library("limma").58 278.165e-99 2488 32466_at 12. and that of B2 µ2 .c("B".08 12.which(ALL$BT %in% c("B". design.64 12. package = "ALL") allB <.ALL[.42 13. data(ALL.matrix(~ 0 + factor(allB$BT)) colnames(design. The type of analysis is speciﬁed by using a factor that deﬁnes the model (design) matrix.11 296.0 3. that of B1 µ1 .4. > cont.adjust.ma Contrasts Levels B .1:5].68 12.

Example 2.262 -7. saveHTML(anntable. Chromosome Location.adjust. The resulting anntable is saved in HTML format in the working directory or the Desktop.handle .551 6.html". UniGene. we have applied a method called “false discovery rate” (fdr) which increases the p-values somewhat in order to reduce the number false positives. PubMed. and communicate various types of results is in the form of an HTML ﬁle.digits=4) > toptabcon <.426 2. Summarizing output in HTML format.contrasts.737e-10 419 1389_at -1.adjust.1:5].ma) fit1 <. It is often desired to combine the typical output from a function like topTable with that of an HTML output page containing various types of information. coef=2.handler functionality.topTable(fit1.5.7852 9. A very convenient manner to summarize.digits=4) ID logFC AveExpr t P.106 8.character(toptabcon$ID).374 5. LocusLink. fit1 <. Cytoband.db".081 1. and Pathway.0976 4. MICRO ARRAY ANALYSIS Observe that the contrast matrix speciﬁes the diﬀerence between the levels B and B1 as well as between B1 and B2.5.019 2. collect. Chromosome.361e-08 7542 37471_at 0.315e-09 6939 36873_at 1.102 CHAPTER 6. coef=2.4890 5.8701 6.db") anntable <. and the aaf. "hgu95av2. It contains a wealth of information aaf. library("annaffy"). the annotation package. Gene Ontology.method="fdr") print(toptabcon[.Value 3389 33358_at 1. The number of genes requested equals 5. GenBank.260 7. The information collected contains the following: Probe. Function.1:5]. "ALLB123. To illustrate this we proceed with the object toptabcon of the previous example.topTable(fit.161e-08 Here.8646 4. Description.method="fdr") > print(toptabcon[.939 7.eBayes(fit1) toptabcon <.library("hgu95av2.303 6.aafTableAnn(as. It can be implemented as follows. title = "B-cell 012 ALL") By the function aafTableAnn various types of information are gathered from the output topTable of the estimated linear model.fit(fit.816e-09 1016 1914_at 2. Symbol. cont. data(ALL.which(ALL$BT %in% c("B". The meaning of the columns can be obtained from the help page of the function.html") hgu95av2. library(GEOquery). analysis of variance (ANOVA). library("annaffy").db).8:9.db". Chromosome location. 1. summaries from Pubmed articles.db") library("ALL"). Example 4. library(hgu95av2. Bioconductor has a useful facility to download publicly available microarray data sets from NCBI. In a similar manner the p-values from the Kruskal-Wallis test can be used to select genes.4. library(limma). KEGG mappings. Example 3. The GDS1365 data contain primed macrophage response to IFN-gamma restimulation after diﬀerent time periods. etc."B1".g. the selected genes can directly be used as input for aafTableAnn. library("multtest").db is a meta data annotation package connecting the requested information by the call to aaf. The purpose is to gain insight into the inﬂuence of IFN-gamma priming on IFN-gamma induced transcriptional responses."B2"))] panova <. 3 and 24 hours and a factor protocol with the levels ”IFN-gamma primed” and ”unprimed”. Using basic R functions. Analyzing public available data. we shall select genes with a signiﬁcant interaction eﬀect.apply(exprs(ALLB). library("hgu95av2. which can be extracted by the function pData.featureNames(ALLB)[panova<0.11:13)]) saveHTML(atab. package = "ALL") ALLB <. aaf. Among the phenotypical covariates of the data there is a factor time with levels 0. "hgu95av2.handler.getGEO("GDS1365") eset <. file="ANOVAonB-cellGroups.6. APPLICATIONS OF LINEAR MODELS 103 on e.000001] atab <. Since researchers are often interested in the interaction between factors.do.g.library(annaffy) gds <. function(x) anova(lm(x ~ ALLB$BT))$Pr[1]) genenames <.aafTableAnn(genenames.ALL[.GDS2eSet(gds. It is also possible to summarize results in an HTML table on the basis of p-values from e. The resulting table is saved as an HTML ﬁle in the working directory (getwd()) or desktop. That is.handler()[c(1:3.log2=T) .

function(x) anova(lm(x ~ prot * time))$Pr[1:3]) pvalt <. data(ALL) > annotation(ALL) [1] "hgu95av2" Hence.pData(eset)$time pval <."mefftime".8:9.db".frame(t(pval)) colnames(pvalt) <.01] atab <.01 & pvalt$mefftime < 0. 1.html") By getGEO the data are downloaded to the disk and next these can be loaded into the R system. the annotation package we need is hgu95av2. file="Two-way ANOVA protocol by time.01 & pvalt$interaction < 0.5 Searching an annotation package Detailed information on microarray experiments is stored in an annotation package.db") [1] "hgu95av2" [4] "hgu95av2_dbInfo" [7] "hgu95av2ALIAS2PROBE" [10] "hgu95av2CHRLOC" "hgu95av2_dbconn" "hgu95av2_dbschema" "hgu95av2CHR" "hgu95av2CHRLOCEND" "hgu95av2_dbfile" "hgu95av2ACCNUM" "hgu95av2CHRLENGTHS" "hgu95av2ENSEMBL" .c("meffprot". We restrict the analysis to the ﬁrst 12625 rows because the additional ones contain not available values. 6.aaf.104 CHAPTER 6."hgu95av2.11:13)]) saveHTML(atab.apply(exprs(eset)[1:12625. MICRO ARRAY ANALYSIS prot <.data.pData(eset)$protocol time <. > library("ALL").].db) > ls("package:hgu95av2.db. The function anova extracts the pvalue of the interaction eﬀect from the estimated linear model. The function pData extracts the factors from the expression set eset. The resulting html ﬁle seems to contain may interesting genes. By GDS2eSet these are transformed to an expression set so that these can be analyzed statistically.handler()[c(1:3. > library(hgu95av2.featureNames(eset)[pvalt$meffprot< 0. Let’s load it and obtain an overview of its functionality.aafTableAnn(genenames."interaction") genenames <.

env = hgu95av2SUMFUNC) [1] NA . > ChrNrOfProbe <. the GenBank accession number. For this we use the get function in order to search an environment for a name. env = hgu95av2GENENAME) [1] "membrane metallo-endopeptidase (neutral endopeptidase. enkephalinase. respectively. env = hgu95av2SYMBOL) [1] "MME" > get("1389_at". CALLA.as.6. brief summaries of functions of the gene products. the Entrez Gene identiﬁer. env = hgu95av2ACCNUM) [1] "J03779" > get("1389_at". > get("1389_at". SEARCHING AN ANNOTATION PACKAGE [13] [16] [19] [22] [25] [28] [31] [34] "hgu95av2ENSEMBL2PROBE" "hgu95av2ENZYME2PROBE" "hgu95av2GO2ALLPROBES" "hgu95av2MAPCOUNTS" "hgu95av2PATH" "hgu95av2PMID" "hgu95av2REFSEQ" "hgu95av2UNIPROT" "hgu95av2ENTREZID" "hgu95av2GENENAME" "hgu95av2GO2PROBE" "hgu95av2OMIM" "hgu95av2PATH2PROBE" "hgu95av2PMID2PROBE" "hgu95av2SYMBOL" 105 "hgu95av2ENZYME" "hgu95av2GO" "hgu95av2MAP" "hgu95av2ORGANISM" "hgu95av2PFAM" "hgu95av2PROSITE" "hgu95av2UNIGENE" The annotation package contains environments with diﬀerent types of information.5. CD10)" > get("1389_at". An easy manner to make the content of an environment available is by converting it into a list and to print part of it to the screen.list(hgu95av2CHR) > ChrNrOfProbe[1] $‘1000_at‘ [1] "16" We recognize the manufacturers identiﬁer of genes and the corresponding chromosome. env = hgu95av2ENTREZID) [1] 4311 > get("1389_at". From these we obtain various types of information on the basis of the manufacturer’s identiﬁer such as "1389_at". gene name. Below we obtain. and the UniGene identiﬁer. Asking information by ?hgu95av2CHR reveals that it is an environment (hash table) which provides mappings between identiﬁers and chromosomes. the gene abbreviation. library(annotate). env = hgu95av2MAP) [1] "3q25. ll1<-GOENTREZID2GO[["4121"]] 6.106 CHAPTER 6.2" Hence. > library(hgu95av2.disp="browser") Another possibility is to collect a list containing PubMed ID. e. env = hgu95av2CHRLOC) 3 3 3 156280152 156280327 156280748 Its cytoband location can also be obtained. journal.type="uid") Obviously.disp="data". data(ALL) > pmid <. and. speciﬁcally. 4121. probes correspond to genes and frequently we are interested in their chromosome location.307734" Let’s use the GenBank accession number to search its nucleotide data base.g. authors. env = hgu95av2UNIGENE) [1] "Hs. MICRO ARRAY ANALYSIS > get("1389_at". > genbank(1430782. available the corresponding GO terms can be obtained and stored in a list. > get("1389_at". in starting position(s).disp="browser") From this we obtain the corresponding GI:179833 number. abstract text.db).6 Using annotation to search literature Given the manufactures probe identiﬁer it is possible to search literature by collecting Pubmed ID’s and to use these to collect relevant articles. library(ALL). > get("1389_at". which can be used to obtain a complete XML document. and publication date.get("1389_at". .1-q25. title. In case we have a LocusLink ID. > library(annotate) > genbank("J03779". we see that the gene is on Chromosome 3 at q arm band 25 subband 1 and 2.env=hgu95av2PMID) > pubmed(pmid. html") 107 6. By changing GOID into Ontology more speciﬁc information pertaining to ontology is extracted. or “ribosome”. "hgu95av2") > pm. SEARCHING GO NUMBERS AND EVIDENCE > absts <. ne <. From the annotate package we may now select the GO numbers which are related to a biological process.pm.lapply(go1389. env = hgu95av2GO) > idl <. The gene ontology consortium deﬁnes three ontologies: A Molecular Function (MF) describes a phenomenon at the biochemical level such as “enzyme”. or “ligand”.6.get("1389_at".abstGrep("neutral endopeptidase". > pmAbst2HTML(absts[[1]]. > library(annotate) > getOntology(go1389. “nucleus”.7 Searching GO numbers and evidence By the phrase “ontology” we mean a structured language about some conceptual domain.function(x) x$GOID) > idl[[1]] [1] "GO:0006508" The list idl contains 8 members of which only the ﬁrst is printed to the screen. From the latter we extract a list and use an apply type of function to extract another list containing GO identiﬁcation numbers.absts[[1]]) Another possibility is to construct an HTML table with the titles.filename="pmon1389_at. To ﬁnd GO numbers and their dependencies we use get to extract a list from the annotation ﬁles hgu95av2GO for example. Each term is identiﬁed by a unique GO number.7.getabst("1389_at". > go1389 <. A Cellular Component (CC) is a unit within a part of the cell such as “chromosome”.pm."BP") [1] "GO:0006508" "GO:0007267" .titles(absts) The list can obviously be searched for regular expressions. “transporter”. A Biological Process (BP) may coordinate various related molecular functions such as “DNA replication” or “signal transduction”.

6.function(x) x$Ontology) We shall use this list in the below. There are functions to obtain parents and children from a GO identiﬁer. MICRO ARRAY ANALYSIS There are various types of evidence such as: inferred from genetic interaction (IGI). > GOMFPARENTS$"GO:0003700" isa isa "GO:0003677" "GO:0030528" > GOMFCHILDREN$"GO:0003700" isa "GO:0003705" . Per GO identiﬁer the type of evidence can be obtained.subset(go1389. Example 1. et.function(x) x$Evidence) > sapply(go1389TAS. > getEvidence(go1389) GO:0004245 GO:0005886 GO:0005887 GO:0006508 GO:0007267 GO:0008237 GO:0008270 "IEA" "TAS" "TAS" "TAS" "TAS" "TAS" "IEA" GO:0046872 "IEA" When we now want to select the GO numbers with evidence of a traceable author statement we can use the subset function to create a list. go1389TAS <.108 CHAPTER 6.getEvidence(go1389)=="TAS") A manner to extract information from this list is by using an apply type of function. inferred from electronic annotation (IEA). > sapply(go1389TAS.8 GO parents and children The term “transmembrane receptor protein-tyrosine kinase” is more speciﬁc and therefore a ’child’ of the more general term parent term “transmembrane receptor” (Gentleman. al. Collecting GO information.function(x) x$GOID) > sapply(go1389TAS. traceable author statement (TAS). 2005). etc. getOntology(go1389. you may end up with many genes. Filter gene by a term.sapply(gC.9 Gene ﬁltering by a biological term An application of working with GO numbers is to ﬁlter for genes which are related to a biological term. go1389 <.c(gonr.mget(pa. and next to transform these to probes.function(x) x$Children) gonrc <.sapply(gP.getGOChildren(gonr) gPC <. Example 1.gC) pa <. to ﬁnd the GO identiﬁers of the biological process. A research strategy may be to start with a probe number. "BP") gP <. . library(GO). library(annotate).sapply(gP. to obtains its parents.function(x) x$Parents) probes <.getGOParents(gonr) pa <.hgu95av2GO2ALLPROBES) probeNames <. env = hgu95av2GO) gonr <. env = hgu95av2GO) gonr <. "BP") gP <. parents. Probe selection by GO.unlist(probes) ALLpr <. 6.c(gonr.unlist(pa). useful for further analysis. data(ALL) go1389 <. GENE FILTERING BY A BIOLOGICAL TERM 109 In case of a list of GO identiﬁers you may want to collect the ontology.unlist(ch)) Example 2.getOntology(go1389.function(x) x$Parents) ch <.6.] > dim(exprs(ALLpr)) [1] 7745 128 Indeed.get("1389_at". and children identiﬁers in a vector.ALL[probeNames. library("ALL"). From a biological point of view it is most interesting to select genes which are related to a certain biological process to be speciﬁed by a term such as ”transcriptional repression”.9.getGOParents(gonr) gC <.gP.get("1389_at".

123) to collect appropriate GO numbers from the environment GOTERM. In particular.c(tran1.db") GOTerm2Tag <.] The GO translated probe names are intersected with the row names of the data giving the logical variable inboth. tran1 <..hgu95av2GO2ALLPROBES$"GO:0016564" tran2 <.tran3) inboth <.eapply(GOTERM. A precaution is taken to select only those names which are not empty. x@Term.sapply(GTL. First we deﬁne a function (Gentleman.tran2. MICRO ARRAY ANALYSIS We combine this with the previous ﬁlter. Next. gene ids for which inboth equals TRUE are selected and the corresponding data are collected in the data frame ALLtran. By dim(exprs(ALLtran)) it can be observed that 26 genes which passed the normality ﬁlter are related to ”transcriptional repression”. library("GO"). 2005. This can be obtained by annotation(ALL).ALLs[tran[inboth]. p. 6.110 CHAPTER 6.hgu95av2GO2ALLPROBES$"GO:0017053" tran <. length) names(GTL[Gl>0]) } > GOTerm2Tag("transcriptional repressor") [1] "GO:0016564" "GO:0016565" "GO:0016566" "GO:0017053" The functions eapply and sapply search an environment like GOTERM by grep for matches of a speciﬁed term.function(term) { GTL <. library("hgu95av2. function(x) {grep(term. More information can be obtained by GOTERM$"GO:0016564. et al. For this we need the annotation package used in the stage of data collection.names(exprs(ALLs)) ALLtran <. This gives the GO terms which can now be translated to probe of the ALLs data.hgu95av2GO2ALLPROBES$"GO:0016566" tran3 <.tran %in% row. library("annotate").10 Signiﬁcance per chromosome After a statistical analysis to ﬁlter and order genes it is often quite useful to do post analysis on the results. The variable tran[inboth] gives the ids by which genes can be selected. after collecting p-values from . value=TRUE)}) Gl <.

sum(rawp[AffimIDChr]<0. Per chromosome it can be tested whether the odds ratio diﬀers from 1 or. the number of remaining signiﬁcant probes.db") rawp <.2] [1.6.2] print(f) [.featureNames(ALL) f <.] 106 638 [2.748559 sample estimates: odds ratio 2.757949 2.1] .2] <. SIGNIFICANCE PER CHROMOSOME 111 a t-test one may wonder whether genes with signiﬁcant p-values occur more often within a certain chromosome.names(xx[xx=="19"]) names(rawp) <. Example 1.f[1. function(x) t.] 832 11049 > fisher. data(ALL). whether there is independence.sum(rawp[AffimIDChr]>0.1. To test for such over or under representation the Fisher test is very useful (see Section 4. and the number of remaining non-signiﬁcant probes.05) f[2.f[1.1] <. The data for the test consist of the number of signiﬁcant probes on Chromosome 19.value) xx <. the number of non-signiﬁcant probes on Chromosome 19.sum(rawp>0.apply(exprs(ALL).matrix(NA. > > > > > > > > > library("ALL"). 1. library("hgu95av2.list(hgu95av2CHR) AffimIDChr <.sum(rawp<0. On the expression values of the ALL data we perform a two sample t-test using the patient group for which remission was achieved and for which it was not achieved.2] <.10. f[2. f[1.1] [.test(x ~ ALL$remission)$p.332e-11 alternative hypothesis: true odds ratio is not equal to 1 95 percent confidence interval: 1.05) .7).2.05).test(f) Fisher’s Exact Test for Count Data data: f p-value = 4.05) .206211 . equivalently.as.1] <.2) f[1.

11 Overview and concluding remarks Many examples are given on using analysis of variance or T -tests for selecting genes with large experimental eﬀects on diﬀerent patient groups. (b) Construct the design matrix and an appropriate contrast matrix.test(f) CHAPTER 6. B1. 6. MICRO ARRAY ANALYSIS Pearson’s Chi-squared test with Yates’ continuity correction data: f X-squared = 52.112 > chisq. ”B3”. Gene ﬁltering on normality per group of B-cell ALL patients. (a) Construct a data frame containing the expression values for the B-cell ALL patients in stage B. Analysis of gene expressions of B-cell ALL patients using Limma.”B3”. (b) How many pass the ﬁlter? (c) Compute a Venn diagram for group ”B2”. df = 1. plot it. The above statistical methods seem to cover the majority of problems occurring in practice. 2.12 Exercises 1. and ”B4”. and give a correct interpretation for each number. The hypothesis of independence is rejected by both tests. p-value = 4. 6. B3.573e-13 The number of signiﬁcant probes is larger for Chromosome 19 resulting in an odds ratio of 2. (d) Collect information on the twenty best gene s in an HTML page.”B2”. B2. .2. B4 from the ALL data. (a) Use genefilter to program the Shapiro normality test separately for each gene of the groups ”B1”. (c) Compute the twenty best genes by topTable.3803.”B4”.

Use grep to ﬁnd the row number of gene 1389_at. Remission achieved. (c) Give the aﬀymetrix names (symbols) of the genes the pass the selection criterion of p-value smaller than 0.names or featureNames. (d) Use the latter to ﬁnd the biological names. excluding not available data. Hint: Use row.12. (e) How many oncogenes are there is total? . (b) Program the two-sample t-test not assuming equal variances to select genes with p-values smaller than 0. The variable ALL$CR has values CR (became healthy) and REF (did not respond to therapy. (e) Is the famous protein p53 is among them? (f) How many unique gene names are there? 5. Give the code. (a) How many persons are classiﬁed as CR and REF. (c) Collect and give the manufactures probe names of the genes with p-values smaller than 0. Remission (genezing) from acute lymphocytic leukemia (ALL). (a) Construct a separate data frame consisting of only those gene expression values from patients that have values CR or REF.001. Hint: You may have to select the persons with values on remission. For the ALL data from its ALL library the patients are checked for achieving remission. respectively.0001 from the twosample T -test not assuming equal variances? Hint: Use the apply functionality to program the test. EXERCISES 113 3. With respect to the ALL data from the ALL library there is a phenotypical variable called remission indicating complete remission CR or refractory REF meaning improvement from disease and less or no improvement. Finding a row number.001.0001.6. (d) Use the probe names to ﬁnd the corresponding gene names. respectively? Hint: Use pData to extract a data frame with phenotypical data. remain ill). 4. (b) How many genes have a p-value smaller than 0. Gene ﬁltering of ALL data. (a) Select the persons with T-cell leukemia which are in stage B1. Stages of B-cell ALL in the ALL data. 6. You may use the function ”table” to ﬁnd the frequencies of the patient types and leukemia stages. Here we meet a missing data problem. The persons with T-cell leukemia which are in stage T2 and T3 can be selected by the variable ALL$BT.na(x)) in apply on the rows to count the number of missing values per row. Use the function function(x) sum(is. (d) For how many genes is the null-hypothesis to be rejected? 8. Select the rows without missing value to perform a two-sample t-test with the . (a) Program a gene ﬁlter step separately for T2 and T3 patients such that only those genes pass which are normally distributed. The data are in the library called ”ALL”. (c) Perform analysis of variance to test the hypothesis of equal population means. To answer the questions below functions from the library ”geneﬁlter” are helpful. B3. Use the Benjamini & Hochberg (1995) (”BH”) adjustment method for the false discovery rate and topTable to report the ﬁve best genes. (a) Download GDS486 and transform it into eset form. (b) What type of contrast matrix would you like to suggest in this situation? Give its code. (c) How many genes pass all ﬁlter steps? (d) How many genes pass normality? 7. Analysis of public micro array data on rheumatoid arthritis.114 CHAPTER 6. (b) Program a second ﬁlter step which passes only those genes with a signiﬁcant p-value from the two sample T -test. A manner to solve it is as follows. B2. Use the limma package to answer the questions below. and B4. MICRO ARRAY ANALYSIS (f) Do the Fisher test on the number of oncogenes out of the total versus the number of signiﬁcant oncogenes out of the selected.

(d) Compute the symbols of the twenty best genes in the sense of having smallest summed p-values. Hint: Re-order the columns into ”NEG”. and between NEG and BCR/ABL.12.biol with values ALL1/AF4. (a) Select the patients on the covariate mol. Does p53 play a role in the path way of the best gene? 9. (b) Collect the ANOVA p-values with contrast between NEG and ALL1/AF4. Report the number of signiﬁcant aﬀy ID’s and the total. (c) Download GDS2126 and repeat the above using ANOVA p-values with the covariate disease. EXERCISES 115 groups in cell.state to indicate the groups. and NEG. Analysis of genes from a GO search.line. BCR/ABL.6. ”ALL1/AF4”. (b) Download GDS711 and repeat the above using ANOVA p-values with the covariate disease. (e) Perform Fisher exact to test the odds ratio equal to one hypothesis. (e) Summarize information of the twenty best genes in an HTML table.state to indicate the groups. and ”BCR/ABL”. Overwrite the vector with the number of missing values with the p-values in a suitable manner. (d) Select the aﬀy ID’s corresponding to the GO ID’s and report its number and the number of signiﬁcant genes. . (c) Find the GO ID’s refereing to the term ”protein-tyrosine kinase” since it mediates many steps due to BCR/ABL translocation.

116

CHAPTER 6. MICRO ARRAY ANALYSIS

Chapter 7 Cluster Analysis and Trees
Given the expression values of several genes, a problem which often arises is to ﬁnd genes which are similar or close. Genes with expressions in small distance may have similar functions and may be potentially interesting for further research. In order to discover genes which form a group there are several methods developed called cluster analysis. These methods are based on a distance function and an algorithm to join data points to clusters. The socalled single linkage cluster analysis is intuitively appealing and often applied in bioinformatics. By this method several clusters of genes can be discovered without specifying the number of clusters on beforehand. The latter is necessary for another method called k-means cluster analysis. Each analysis produces a tree which represents similar genes as close leaves and dissimilar ones on diﬀerent edges. An other measure to investigate similarity or dependency of pairs of gene expressions is the correlation coeﬃcient. Various examples of applications will be given. It prepares the way to searching a data set for directions of large variance. That is, since gene expression data sets tend to be large, it is of importance to have a method available which discovers important “directions” in the data. A frequently used method to ﬁnd such directions is that by principal components analysis. Its basic properties will be explained as well as how it can be applied in combination with cluster analysis. In applications where it is diﬃcult to formulate distributional assumptions of the statistic it may still be of importance to construct a conﬁdence interval. It will be illustrated by several examples how the bootstrap can be applied to construct 95% conﬁdence intervals. Many examples are given to clarify the application of cluster analysis and principal components analysis. 117

118

CHAPTER 7. CLUSTER ANALYSIS AND TREES

7.1

Distance

The concept of distance plays a crucial role in all types of cluster analysis. For real numbers a and b a distance function d is deﬁned as the absolute value of their diﬀerence d(a, b) = |a − b| = (a − b)2 .

The properties of a distance function should be in line with our intuition. That is, if a = b, then d(a, a) = 0 and if a = b, then d(a, b) > 0. Hence, the distance measure should be deﬁnitive in the sense that d(a, b) = 0 if and only if a = b. Since the square is symmetric, it follows that d(a, b) = |a − b| = (a − b)2 = (b − a)2 = |b − a| = d(b, a).

In other words, d(a, b) = d(b, a), the distance between a and b equals that between b and a. Furthermore, it holds for all points c between a and b that d(a, b) = d(a, c) + d(c, b). For all points c not between a and b, it follows that d(a, b) < d(a, c) + d(c, b). The latter two notions can be summarized by the so-called triangle inequality. That is, for all real c it holds that d(a, b) ≤ d(a, c) + d(c, b). Directly going from a to b is shorter than via c. Finally, the distance between two points a and b should increase as these move further apart. Example 1. Let a = 1 and b = 3. Then, obviously, the distance d(1, 3) = 2. The number c = 2 is between a and b, so that d(1, 3) = 2 = 1 + 1 = d(1, 2) + d(2, 3) and the triangle inequality becomes an equality. For the situation where gene expression values for several patients are available, it is of importance to deﬁne a distance for vectors of gene expressions such as a = (a1 , · · · , an ) and b = (b1 , · · · , bn ). We shall concentrate mainly on the Euclidian distance, which is deﬁned as the root of the sum of the squared diﬀerences
n

d(a, b) =
i=1

(ai − bi )2 .

7.1. DISTANCE

119

The distance measure satisﬁes the above properties of deﬁniteness, symmetry, and triangle inequality. Although many other, but often highly similar, distance functions are available we shall mainly concentrate on Euclidian distance because it is applied most frequently in bioinformatics. Example 2. Suppose that a = (a1 , a2 ) = (1, 1) and b = (b1 , b2 ) = (4, 5). Then √ d(a, b) = (a1 − b1 )2 + (a2 − b2 )2 = (1 − 4)2 + (1 − 5)2 = 9 + 16 = 5. Since the diﬀerences are squared it is immediate that d(a, b) = d(b, a), the distance from a to b equals that √ from b to a. For c = (c1 , c2 ) = (2, 2) we √ √ have that d(a, c) = 2, d(b, c) = 22 + 32 = 13. Hence, √ √ d(a, b) = 5 < 2 + 13 = d(a, c) + d(b, c), so that the triangle inequality is strict. This is in line with our intuitive idea that the road directly from a to b is shorter than from a to b via c. Example 3. To compute the Euclidian distance between two vectors one may use the following. > a <- c(1,1); b <- c(4,5) > sqrt(sum((a-b)^2)) [1] 5 Example 4. Distances between Cyclin gene expressions. By the build-infunction dist the Euclidian distance between two vectors of gene expression values can be computed. To select genes related to the biological term ”Cyclin” and to compute the Euclidian distance between the gene expression values of the Golub et al. (1999) data, we may use the following. > library(multtest); data(golub) > index <- grep("Cyclin",golub.gnames[,2]) > golub.gnames[index,2] [1] "CCND2 Cyclin D2" [2] "CDK2 Cyclin-dependent kinase 2" [3] "CCND3 Cyclin D3" [4] "CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1)" [5] "CCNH Cyclin H"

120

CHAPTER 7. CLUSTER ANALYSIS AND TREES

[6] "Cyclin-dependent kinase 4 (CDK4) gene" [7] "Cyclin G2 mRNA" [8] "Cyclin A1 mRNA" [9] "Cyclin-selective ubiquitin carrier protein mRNA" [10] "CDK6 Cyclin-dependent kinase 6" [11] "Cyclin G1 mRNA" [12] "CCNF Cyclin F" > dist.cyclin <- dist(golub[index,],method="euclidian") > diam <- as.matrix(dist.cyclin) > rownames(diam) <- colnames(diam) <- golub.gnames[index,3] > diam[1:5,1:5] D13639_at M68520_at M92287_at U09579_at U11791_at D13639_at 0.000000 8.821806 11.55349 10.056814 8.669112 M68520_at 8.821806 0.000000 11.70156 5.931260 2.934802 M92287_at 11.553494 11.701562 0.00000 11.991333 11.900558 U09579_at 10.056814 5.931260 11.99133 0.000000 5.698232 U11791_at 8.669112 2.934802 11.90056 5.698232 0.000000 By the grep function the order numbers of the genes with the phrase ”Cyclin” in their names are assigned to the vector called index. The euclidian distances are assigned to the matrix called diam. Its diagonal has distances between identical genes which are, of course, zero. The distance between the ﬁrst (CCND2 Cyclin D2) and the third (CCND3 Cyclin D3) is relatively small, which is in line with the fact the these genes have related functions. Note, however, that there are genes with in smaller distance. Example 5. Finding the ten closest genes to a given one. After selecting certain genes it often happens that one wants to ﬁnd genes which are close to the selected ones. This can be done with the genefinder functionality by specifying either an index or a name (consistent with the geneNames of the exprSet). To ﬁnd genes from the ALL data (Chiaretti et al., 2004) close to the MME expression values of the probe with identiﬁer 1389_at, we may use the following. library("genefilter"); library("ALL"); data(ALL) closeto1389_at <- genefinder(ALL, "1389_at", 10, method = "euc") closeto1389_at[[1]]$indices round(closeto1389_at[[1]]$dists,1) featureNames(ALL)[closeto1389_at[[1]]$indices] 1.1 If desired.2. It computes the the distances between the genes and performs a single linkage cluster analysis.2). 7. 1). names <.j and xj in J} ."g5")."g4". and g 5 = (5. the nearest two are determined and these are merged into one cluster. An explanatory example. from two persons.list(c("g1". The expressions for each gene can be seen as coordinates on two perpendicular axis p1 and p2 . Next. see Chapter 6 of the manual ”An Introduction to R”. ) : xi in I i. g 4 = (3.2. The algorithm of single linkage cluster analysis starts with creating as many clusters as data points. 5). To illustrate single linkage cluster analysis suppose we the following ﬁve gene expressions g 1 = (1. Hence. From the output it can be observed that the gene expressions of row 2653 with probe identiﬁer 32629_f_at has the smallest distance (12.1 Single Linkage A cluster I is simply a set of data points I = {xi }.2).6) to those of 1389_at. Then the next two nearest clusters are determined and merged into one cluster. J) = min {d(xi . TWO TYPES OF CLUSTER ANALYSIS 121 The function genefilter produces a list from which the selected row numbers can be extracted as well as the probe names can be found."g2".1 which illustrates the idea.2 Two types of Cluster Analysis Some important types of cluster analysis are deﬁned and illustrated here. This process continuous until all points belong to one cluster. In single linkage cluster analysis the distance between clusters I and J is deﬁned as the smallest distance over all pairs of points of the two clusters: d(I. Example 1. xj . 7."g3"."p2")) 1 For information on lists. . 2).7. these can be used for further analysis. the distance between the two clusters is the same as that of the nearest neighbors. The script below produces Figure 7. g 3 = (3. 2. where xi is the i-th vector with gene expressions. g 2 = (1.c("p1". 2. corresponding to the horizontal line at height x2 x4 . say I = {g 1 .3.method="euclidian").0 0.dat.122 CHAPTER 7. g 2 }. the minimal distance between clusters can be read from the Euclidian distance matrix.36 > sl.out<-hclust(dist(sl.dat. g 2 ) = 0.5 3.6)) text(sl.10 in the tree.0 x4 x3 2 1. g 4 ) = 0.66 5.2 this is illustrated by the horizontal line at height 0.10.5. CLUSTER ANALYSIS AND TREES Cluster Dendrogram x5 5 3.digits=3) x1 x2 x3 x4 x2 0.2: Tree of single linkage cluster analysis.1.10 x3 2. Next.clus. "single") Figure 7.dat. ylim=c(0.dimnames = names) plot(sl.33 0. having d(g 1 .30 x5 5. g 4 }.30.0 4 Height 0.labels=row.39 2.dat <.matrix(c(1. g 4 . method = "euclidian") hclust (*.clus.3.61 3. Then the nearest two points (genes) from the Euclidian distance matrix are g 1 and g 2 .5 2.1: Plot of ﬁve points to be clustered. the new cluster J = {g 3 .0 2.clus.19 x4 2. Since the smallest is d(g 3 .24 2.clus. byrow = TRUE.6). xlim=c(0.dat.5 x5 a2 3 x3 x1 x2 x1 1 2 3 a1 4 5 1 dist(sl.3.method="single") > plot(sl.ncol = 2. Figure 7.1.clus.names(sl.dat)) > print(dist(sl.clus. g 5 are seen as three diﬀerent clusters. The other three data points g 3 .out) At the start each data point is seen as a separate cluster.2.dat. sl.1.5).method="euclidian").5 1. In Figure 7.clus. These two data points are merged into one cluster.1.type="n".59 3. "single") 10 17 11 16 19 25 28 30 24 21 29 Figure 7. sl.8 0.method="euclidian").6 Height 6 12 9 0. Finally. 0.4: Three clusters with different standard deviations.out) From the resulting tree in Figure 7. Figure 7.0.1). TWO TYPES OF CLUSTER ANALYSIS 123 0. see the corresponding horizontal line at this height. J. If. I. it can be observed that the distance between cluster I and J is 2. Example 2. however.3 one might get the impression that there are ﬁve separate clusters in the data. Hence.30. and the data point g 5 equals d(g 4 .7.2 0. Now there are three clusters. Note. the distance between cluster {g 1 .19. and K = {x5 }.0 0.3: Example of three without clusters. It is of importance to have some experience with data that does and does not contain clusters. method = "euclidian") hclust (*. From the Euclidian distance matrix. g 5 ) = 3. Relating data generation processes to cluster trees. however. 1).0 0 7 13 22 4 1 2 3 4 5 dist(rnorm(20. then there are diﬀerent processes producing separate clusters.out<-hclust(dist(rnorm(20. the cluster I and J are merged into one.method="single") plot(sl. "single") 3 5 2 1 15 18 8 14 . Cluster Dendrogram Cluster Dendrogram 1. method = "euclidian") hclust (*.2. g 4 }. To illustrate 23 26 27 2 1 9 7 10 5 6 4 3 8 16 19 20 17 15 13 18 12 11 14 dist(x. g 3 . g 2 . the data are generated by diﬀerent normal distributions. see the corresponding horizontal line at this height. that there is no underlying data generation process which produces separate clusters from diﬀerent populations.4 Height 20 0. To illustrate this we perform single linkage cluster analysis on twenty data points from the standard normal population.36. Application to the Golub (1999) data. 0.5 dist(clusdata.1) population.0.0 2. ten from N (3.2 1. Recall that the ﬁrst twenty seven patients belong to ALL and the remaining eleven to AML and that we found earlier that the expression values of the genes ”CCND3 Cyclin D3” and ”Zyxin” diﬀer between the patient groups ALL and AML.5 0.10. it can be observed that there clearly exist three clusters.4.4 0 29 21 2 0. How to produce this plot and a single linkage cluster analysis is shown by the script below. Figure 7.method="single")) plot(sl.5).1.0. Figure 7.8 35 0.5 1.0 1 Height Zyxin 0. Cluster Dendrogram ALL AML 1. 0.out) From the tree in Figure 7.3. x <.rnorm(10. 1). ten data points were sampled from the N (0. "single") CCND3 Cyclin D3 Figure 7.5 illustrates that the patient groups diﬀer with respect to gene expression values.5 2. Example 3. 1 3 6 11 23 8 27 7 9 37 32 36 38 33 28 31 −1 0.124 CHAPTER 7.c(rnorm(10.5: Plot of gene ”CCND3 Cyclin D3” and ”Zyxin” expressions for ALL and AML patients.6 0.2 26 4 13 15 22 24 25 16 20 10 19 5 12 −0.0 0.6: Single linkage cluster diagram from gene ”CCND3 Cyclin D3” and ”Zyxin” expressions values.1).0.0)) plot(hclust(dist(x. but that some caution is indeed in order.method="euclidian").0 1.5).0 17 18 2 14 34 30 . method = "euclidian") hclust (*. These examples illustrate that results from cluster analysis may very well reveal population properties.rnorm(10. CLUSTER ANALYSIS AND TREES this. and ten from N (10. The latter yields new clusters of which the means are calculated (step 1). When the data points are independent and identically distributed. ak ) k=1 i∈Ik over all possible points a1 . . then the cluster means converge in probability to the corresponding population means (Pollard.6 gives the tree from single linkage cluster analysis. package="multtest") clusdata <. · · · .method="single")) Figure 7.numeric(gol.data. xn the method seeks to minimize the function K nk d2 (xi . Apart from three expressions the tree shows two clusters corresponding to the two patient groups.cl. It then computes the cluster means (step 1) and constructs a new partition by associating each point with the closest cluster mean (step 2).2. This is accomplished by an algorithm (Hartigan & Wong.]. The latter occurs when the data points no longer change clusters."AML")) plot(clusdata. These two steps are repeated until convergence.method="euclidian"). It is deﬁned by minimizing the within cluster sum of squares over K clusters. TWO TYPES OF CLUSTER ANALYSIS 125 data(golub. given the data points x1 .fac <."Zyxin") gol. 1981).7.levels=0:1. pch=as. 1979) which starts by partitioning the data points into K initial clusters. either at random or using some heuristic device.2 k-means K-means cluster analysis is a popular method in bioinfomatics."AML"). The iterative algorithm is fast in the sense that it often converges in less iterations than the number of points n. labels= c("ALL". it holds that these are equal to the mean per cluster. 7.]) colnames(clusdata)<-c("CCND3 Cyclin D3". For the optimal points a1 .pch=1:2) plot(hclust(dist(clusdata.factor(golub.frame(golub[1042.2.golub[2124. · · · aK . that is ak = xk for each cluster k. · · · aK . That is. Then it constructs a new partition by associating each point with the closest cluster mean (step 2).legend=c("ALL". but it need not to attain the global minimum.fac)) legend("topright". clus.1] 2 3 dist(sl.1] [. 2) K-means clustering with 2 clusters of sizes 50. To illustrate k-means cluster analysis we shall simulate gene expressions from two diﬀerent normal populations. + matrix(rnorm(100.0 Height 0.01940342 2 0. CLUSTER ANALYSIS AND TREES Cluster Dendrogram 3. 0.5 3 3. Relating a data generation process to k-means cluster analysis.5 1. 50 Cluster means: [.0 0 1. method = "euclidian") hclust (*. "single") Figure 7. > data <.2] 1 1.5 data[.7: K-means cluster analysis.0. The data points are collected in two matrices of order ﬁfty by two which are placed one above the other.rbind(matrix(rnorm(100. we randomly take ﬁfty gene expressions for two persons from the N (0.07320413 Clustering vector: [1] 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 x2 x4 .8: Tree of single linkage cluster analysis.2] 1 2 x5 x3 −1 x1 −1 0 1 data[.126 CHAPTER 7. ncol = 2)) > cl <.01720177 0.0.0 0.87304978 2.5) population.2.5) population and ﬁfty expressions for two persons from the N (2.5).5 2.kmeans(data. 0.0 2.dat. That is. ncol = 2).5). Example 1. On the total of one hundred data points a (k =)2-means cluster analysis is performed. Figure 7.0. 0) and (2. > initial <. as follows. Another possibility is to use rational initial starting values for the cluster means. 1979) can be used to estimate 95% conﬁdence intervals around cluster means. To cope with the danger of suboptimal solutions one may simply run the algorithm repeatedly by using the nstart option. nstart = 10) The so-called bootstrap (Efron.kmeans(data.0. see Figure 7. nrow = 2.60733 20. The sum of the within cluster sum of squares equals the minimal function value obtained by the algorithm. byrow=TRUE) > cl<. The variable cl$cluster contains cluster membership and can be used to specify the color of each data point a plot. If the number of clusters is not at all clear. . initial.54411 Available components: [1] "cluster" "centers" "withinss" "size" The output of k-means cluster analysis is assigned to a list called cl. the sample means of potential clusters or the hypothesized population means can be used.7.2. In particular.matrix(c(0. Before performing a k-means cluster analysis a plot from a single linkage cluster analysis may reveal the number of clusters. ncol=2. The Clustering vector indicates to which cluster each data point (gene) belongs and that these correspond exactly to the two populations from which the data are sampled. cex=2) The data points are plotted in red and black circles and the cluster means by a star. col = 1:2. pch = 8. For cases where the number of clusters is only moderately clear. > plot(data. The idea is to re-sample with replacement from the given sample one thousand times with replacement and to compute quantiles for the corresponding conﬁdence intervals. Such solutions are of limited scientiﬁc value. col = cl$cluster) > points(cl$centers.2.2). Observe that the cluster means are fairly close to the population means (0. the algorithm is more sensible to get stuck into a solution which is only locally optimal. 2). then it becomes questionable whether k-means is appropriate.7. TWO TYPES OF CLUSTER ANALYSIS 127 [38] 2 2 2 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 [75] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Within cluster sum of squares by cluster: [1] 22.

0.3]. Application to the Golub (1999) data.nrow=nboot.cl <.1098886 0.975)) 2.0.5% 97.5% 1.].730495 2. 2) are accepted.5% 97.kmeans(data.frame(golub[1042.009014 > quantile(boot. nboot<-1000 boot.5866682 1.1].replace=TRUE).1627979 > quantile(boot.]) } > quantile(boot.]. a 2-means cluster analysis of these gene expression values is appropriate here. initial.c(0.star <.c(0.]) > colnames(data)<-c("CCND3 Cyclin D3".025.nstart = 10) > cl K-means clustering with 2 clusters of sizes 11.128 CHAPTER 7.cl[."Zyxin") > cl <. Hence.2].025.5% 97.cl$centers[2.kmeans(dat.6355909 1. nstart = 10) boot.5% -0.2947926 .0.star.golub[2124.19721732 > quantile(boot.cl[.162019 From the bootstrap conﬁdence intervals the null hypothesis that the cluster population means are equal to (0.c(0. In the above we found that the expression values of the genes ”CCND3 Cyclin D3” and ”Zyxin” are closely related to the distinction between ALL and AML.cl[.c(cl$centers[1.975)) 2.100.matrix(0.8938826 -0. 2.] cl <.data[sample(1:n. CLUSTER ANALYSIS AND TREES n <.c(0.025.975)) 2.898407 2. Example 2.5% -0.975)) 2.ncol = 4) for (i in 1:nboot){ dat. > data <.025. 0) and (2.] <.cl[i.4].data.5% 1.cl[. 27 Cluster CCND3 1 2 means: Cyclin D3 Zyxin 0.0.04830563 0.5% 97.

5% 0.025.2.1].2548907 0.c(0.cl)) X1 X2 X3 X4 0.0.0.02420802 The diﬀerence between the bootstrap means and the k-means from the original data gives an estimate of the estimation bias.c(0. The estimation is quite precise because the 95% bootstrap conﬁdence intervals are fairly small.025.9.5% -0.259608 1.975)) 2.025. .cl[.5% 97.5% 97. It can be observed that the bias is small.692813 2.842225 The two clusters discriminate exactly the ALL patients from the AML patients.2]. and the ALL patients are in red and the AML patients in black. TWO TYPES OF CLUSTER ANALYSIS 129 Clustering vector: 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 27 28 29 30 31 32 33 34 35 36 37 38 2 1 1 1 1 1 1 1 1 1 1 1 Within cluster sum of squares by cluster: [1] 4.6381860 1.c(0.0. where expression values of CCND3 Cyclin D3 are depicted on the horizontal axis and those of Zyxin on the vertical.800581 > quantile(boot.8945878 -0.092361 > quantile(boot.9835898 > quantile(boot.0.975)) 2. By the bootstrap the cluster means and their conﬁdence intervals can be estimated.60802142 -0.5% 97.cl[.7.frame(boot.3].cl[.cl[.975)) 2.2989426 > quantile(boot.025.975)) 2. > mean(data.733248 19.5707477 1. This can also be seen from Figure 7.5% 97.5% 1.4].5% 1.c(0.

0 0.5 1.130 CHAPTER 7. CLUSTER ANALYSIS AND TREES Zyxin −1 0 1 2 −0.9: Plot of kmeans (stars) cluster analysis on CCND3 Cyclin D3 and Zyxin discriminating between ALL (red) and AML (black) patients.0 1. then the variables are . the correlation coeﬃcient ρ is estimated by ρ= n i=1 (xi n i=1 (xi − xi )(yj − y j ) n i=1 (yj .5 CCND3 Cyclin D3 Figure 7.5 2.0 2. yn ). · · · . For two sequences of gene expressions such as x = (x1 .5 0. If the value is close to either of these values. − xi )2 − y j )2 The value of the correlation coeﬃcient is always between minus one and plus one.3 The correlation coeﬃcient A frequently used coeﬃcient to express the degree of linear relationship between two sets of gene expression values is the correlation coeﬃcient ρ. · · · . xn ) and y = (y1 . 7.

THE CORRELATION COEFFICIENT 131 linearly related in the sense that the ﬁrst is a linear transformation of the second.golub[2289. By moving the slider slowly from the left to the right it can be observed that all points are approximately on a straight line. we ﬁnd its row numbers. then the data points are distributed along contours of circles. Example 2. This gene encodes for highly conserved mini-chromosome maintenance proteins (MCM) which are involved in the initiation of eukaryotic genome replication. it can be observed that the correlation coeﬃcient changes to nearly ±1. Another teaching demonstration. collect the gene expression value in vectors x and y.y). Example 3.3.].examp(1000) of the TeachingDemos package is quite useful. Teaching demonstration. We shall illustrate the correlation coeﬃcient by two sets of expression values of the MCM3 gene of the Golub et al.6376217 The value is positive which means that larger values of x occur together with larger values of y and vice versa. By . (1999) data. the null hypothesis H0 : ρ = 0 can be tested against the alternative H0 : ρ = 0.cor. To develop intuition with respect to the correlation coeﬃcient the function run. This can also be observed by plot(x. When the correlation is near zero. By next adding one outlier.demo() it is possible to add and delete points to a plot which interactively recomputes the value for the correlation coeﬃcient. Here. > library(multtest).golub[2430. and compute the value of the correlation coeﬃcient by the function cor(x. y <. That is. Example 1.] > cor(x. It launches an interactive plot with 1000 data points on two random variables X and Y .points.test. This illustrates that the correlation coeﬃcient is not robust against outliers. data(golub) > x <. By the function put. By the function cor.y). By ﬁrst creating a few points that lie together on a circle the corresponding correlation coeﬃcient will be near zero.7. Application to the Golub (1999) data.y) [1] 0. there are constants a and b such that axi + b = yi for all i. If the sign of the correlation coeﬃcient is positive. then small/large values of X tend to go together with small/large values of Y .

cor(dat.star <.] <.1000. CLUSTER ANALYSIS AND TREES the function cor.1]} > mean(boot.c(0. 1979) is to obtain thousand samples from the original sample with replacement and to compute the correlation coeﬃcient for each of these. we reject the null hypothesis of zero correlation. Another method to construct a 95% conﬁdence interval is by the bootstrap. > cor.test.data[sample(1:nrow(data).cor) [1] 0.y).matrix(c(x. boot.5% 0.975)) 2.ncol = 1) > data <. Conﬁdence interval by the bootstrap.star)[2.1].matrix(0. the null hypothesis H0 : ρ = 0 can be tested against the alternative H0 : ρ = 0.025.7952115 sample estimates: cor 0.replace=TRUE).3993383 0. The left bound of the conﬁdence interval falls far to the right hand side of zero.132 CHAPTER 7.byrow=FALSE) > for (i in 1:nboot){ + dat. p-value = 1. Example 4.test(x.0.] + boot.cor <.cor[.cor[i.6534167 > quantile(boot.ncol=2.2207915 0. This yields thousand coeﬃcients from which the quantiles for the 95% conﬁdence interval can be computed.9204865 . The idea (Efron. It also estimates a 95% conﬁdence interval for ρ.5% 97.y) Pearson’s product-moment correlation data: x and y t = 4. Since the corresponding p-value is very small.6376217 The test is based on the normality assumption and prints therefore a t-value. df = 36. > nboot <.nrow=nboot.9662.666e-05 alternative hypothesis: true correlation is not equal to 0 95 percent confidence interval: 0.

1 .7. Application to the Golub (1999) data. This indicates that the assumption of normality may not be completely valid here. The ALL and AML patients of the Golub et al.test.1. where e. A direction is deﬁned as a linear combination Zk of the data Z by a vector k with weights. The latter contains the correlations between each pair of patients (variables). we reject the nullhypothesis of zero correlation. data(golub) > corgol<. function(x) cor(x.4 Principal Components Analysis To make the basic ideas behind principal components analysis explicit. The whole idea of principal components analysis is to ﬁnd new directions in the data along which there is maximal variation.40 which belongs to the second gene of the ﬁrst patient. Since the conﬁdence interval does not contain zero. The data are collected in a 6 by 2 data matrix Z. PRINCIPAL COMPONENTS ANALYSIS 133 Observe that the 95% conﬁdence interval is larger than that found by cor. The direcj=1 tion of maximal variation is deﬁned as the linear combination with maximal variance.golub. 2007). In particular. (1999).cl)) > o <. 7.4. element z21 is expression value -0. To ﬁnd this direction the correlation matrix plays an important role.order(corgol) By golub.cl. Example 5. A manner to select genes it by the correlation of the expression values with this binary vector..g. it is wise to start with a small artiﬁcial example.gnames[o[3041:3051].2] it can be seen that various of these genes seem indeed to have important cell functions referred to by Golub et al. 1. In our case correlations between the columns (patients) in Table 7. Suppose that for six genes the standardized expression values on two patients (variables) became available as these are given in Table 7. The i-th element of the linear combination is the weighted sum 2 zij kj . > library(multtest). Such can be computed by using the apply functionality. Interleukin 8 is recently related to inﬂammatory cytokine production in myeloid cells (Tessarz et al.apply(golub. (1999) data are indicated by zero and ones of the binary vector golub.

A similar result follows by observing that the diﬀerences per row are equal in absolute value. The crux of principal components analysis is that a linear combination with the same direction as the weights represent the direction of maximum variation.8 1 1 = 1. We shall determine such a constant by ﬁnding the weights vector ﬁrst. Both vectors k and Rk can be plotted in the xy-plane.8 elsewhere.08 −0.8 1 2 1 = 2.1: Data set for principal components analysis.8 1. 2 .6 .63 1.134 CHAPTER 7. To do so observe from our correlations matrix that the sum of both rows equals 1. 1)2 of the sample correlation matrix R. To illustrate a direction let’s try the linear combination k = (2. that is there exists a constant d such that Rk = dk.17 −0.8 2.93 can be placed in a matrix R.0) to the point with x = 2 and y = 1.8 = 1. which has ones on the diagonal and the value 0.22 −0. gene gene gene gene gene gene 1 2 3 4 5 6 Var 1 Var 2 1.8.10. 1) yields Rk = 1 0. 1) is plotted by drawing an arrow from (0. Such is the case if Rk diﬀers from k only by a constant of multiplication.8.8 1 1 1 = 1.8 0.8.97 −1. The vector (2. −1) For the sake of simple notation we shall not use the transposition operator T to indicate rows. This gives Rk = 1 0. CLUSTER ANALYSIS AND TREES Table 7.6) in Figure 7.96 −0.93 0. taking k = (1.8k. That is.40 0.61 −0.79 0. So that we obtain d = 1.38 −1.8 0. It can be observed that the two vectors (arrows) do not fall on the same line and therefore have diﬀerent directions. Taking k = (1. 2. This is done completely similar for (2.

0 0.79. It is convenient to store the data of the ﬁrst two columns of Table 7.8 0. Now the ﬁrst principal component is deﬁned as Zk1 and the second as Zk2 .2 −0. 0. 1. -1.0 0.0 2.2] 2. In practical applications the actual computation of eigenvectors and eigenvalues is performed by well-designed numerical methods (Golub & Van Loan.10: Vectors of linear combinations. −0. Since the length of √ eigenvector (1.2 1 −1 = 0.38.0 Z[.71.1] 1 2 Figure 7. 0.11: First principal component with projections of data.40.1] 2.5 1. 1/ 2) ≈ (0. .5 V[. Eigenvectors are often re-scaled by dividing by their Euclid√ √ ian length. −1) also equals 2 the re-scaled second eigenvector equals √ √ k2 = (1/ 2. we obtain √ √ the new eigenvector k1 = (1/ 2. PRINCIPAL COMPONENTS ANALYSIS yields Rk = 1 0. 3. −1/ 2) ≈ (0.5 −2 −2 −1 0 1 2 −1 0 Z[.0 2. Figure 7. The correlations matrix can be computed by the built-in-function cor and the eigenvectors and eigenvalues by the built-in-function eigen.0 0.5 V[.4.7. Z <.5 3. 1983). 0. Example 1. Since the Euclidian length of (1.2] 0. -0. 0.71).1 as a matrix object called Z. Using R on the above data.matrix(c( 1.2k.63.71).97.0 1. 1) is 12 + 12 = 2.2 = 0.93. as follows.0 1.71.5 1.22.8 1 1 −1 = 0. 135 A vector k for which Rk = dk holds is called an eigenvector corresponding to the eigenvalue d.

eigen(cor(Z)) The output is stored as an object called K which can be printed to the screen in two digits.96. To compute the principal components we use the matrix multiplication operator %*%. scores=TRUE) pca$scores .8 0. for instance.028 [4.11. center = TRUE.74 -0.290 [2.] -1.] -1.1]. digits=2) [.63 and y coordinate 1.17.559 [6.09 0. To print the scores on the ﬁrst and the second principal component one can use the following. > print(Z %*% K$vec.] 1. -0. byrow=TRUE) K <.08.136 CHAPTER 7.] 0. > print(K.] 0.61.93).1] [. Z%*%K$vec[. Gene 1.1] [. has x coordinate 1.princomp(Z. nrow=6. Then the ﬁrst principal component is deﬁned as the linear combination of the data with the ﬁrst eigenvector.] -0.71 The eigenvalues are assigned to K$values and the eigenvectors are the columns of K$vectors. CLUSTER ANALYSIS AND TREES -1.226 To illustrate the ﬁrst principal component the six data points from the Z matrix are plotted as small circles in Figure 7.] 0. pca <.] 2.2$vectors [.34 -0.28 -0.02 0.2] [1.71 [2.digits=2) $values [1] 1. A convenient manner to perform principal components analysis is by using the built-in-function princomp.71 0.841 [3. cor=TRUE.2] [1. -0.80 0. -0.22 and appears therefore in the right upper corner. as follows. -0.71 -0.212 [5. Application to the Golub (1999) data. Then it may be useful to explore simultaneously a two dimensional visualization of the genes and the patients. For this reason.array(dim=c(nboot. Reason for which these will be neglected. The second is the so-called elbow rule saying that when the ﬁrst few eigenvalues are large and the remaining considerably smaller. If there are a few large eigenvalues. the principal components contain less (measurement) error than the individual variables.golub. Example 2. Principal components analysis is a descriptive method to analyze dependencies (correlations) between variables.2484411 1.eigen(cor(dat.0713373 0.0757158 1. The eigenvalues represent an amount of variance related to the component.8 and the second 0.7. cluster analysis on the values on the principal components may be useful.] eigenvalues[i.p)) for (i in 1:nboot){dat. On the basis of the eigenvalues the number of interesting directions in the data can be evaluated by two rules of thumb.replace=TRUE).8/2 = 0. Furthermore. indicating that the persons are dependent to a large extent. nboot<-1000 eigenvalues <.] <. The ﬁrst ﬁve eigenvalues from the correlation matrix of golub can be printed by the following. PRINCIPAL COMPONENTS ANALYSIS 137 The scores are the component scores and the loadings from princomp are the eigenvectors.4.9 or 90% of the variance. The ﬁrst is that each eigenvalue should represent more variance than that of any of the observed variables. n <.nrow(data) . > eigen(cor(golub))$values[1:5] [1] 25. so that the ﬁrst represents 1.7365232 Because the eigenvalues are arranged in decreasing order the sixth to the 38th are smaller than one.star <. data <. then there are equally many directions in the data which summarize the most important variation among the gene expressions. Applying the previous bootstrap methods to estimate 95% conﬁdence intervals for the eigenvalues we obtain the following intervals. it can be rewarding to study the weights of the eigenvectors because these may reveal a structure in the data otherwise gone unnoticed. In the previous example the ﬁrst component has variance 1.4382629 2. Finally. p <.star))$values} .data[sample(1:n. then the ﬁrst few are the most interesting.2.ncol(data). The ﬁrst eigenvalue is by far the largest. 025.6853702 0.as.00646 2 1. reason for which it is neglected. The percentages of variance explained by the ﬁrst two components can be computed by sum(eigen(cor(golub))$values[1:2])/38*100.5 182 2653 −5 1754 1798 2233 2397 . 1910 2874 892 1882 885 849 0. method = "euclidian") hclust (*.j]. The null hypothesis of eigenvalue being equal to one is accepted for the fourth component and rejected for the ﬁrst three and the ﬁfth.258030 3 1.13: Single linkage cluster diagram of selected gene expression values.j].386252 4 0.83581 26. + c(0. It can be checked that all correlations between the patients are positive.12: Scatter plot of selected genes with row labels on the ﬁrst two principal components. Thus the ﬁrst two components represent more than 72% of the variance in the data.0 −10 −10 −5 0 X1 5 10 15 dist(leu.9917813 1.0.5 5 Height X2 893 1.920871 2.145990 1. the data allow for a reduction in dimensions from thirthy eight to two.138 CHAPTER 7. "single") Figure 7. CLUSTER ANALYSIS AND TREES > for (j in 1:p) print(quantile(eigenvalues[. Hence.5% for (j in 1:5) cat(j.025."\n" ) 1 24.4052%.0 2459 1882 892 2289 893 2673 1616 182 849 313 2653 2430 1101 504 316 2397 885 1756 2611 1910 450 2874 2233 2350 1754 1798 1911 2749 1737 2321 792 2761 808 68 2.154291 5 0.975))) 2.numeric(quantile(eigenvalues[.0 2289 0 2459 1.5 2350 1737 1911 2749 2761 792 2321 68 808 2611 450 504 313 1756 2430 316 1616 1101 2673 0.975))).0 2. Cluster Dendrogram 10 3.c(0.0.7995948 The cat function allows for much control in printing. which yields the amount 72. Figure 7. Thus the fourth represents less variance than an individual variable.5% 97.

princomp(golub. Hence. the AML-ALL distinction is discovered by the second component. By contrasting ALL patients with AML patients a second to the largest amount of variance is explained in the data.gnames[o[1:10]. All weights of the ﬁrst eigenvector are positive and have very similar size (all are between 0. The left and bottom axis refer to the component scores and the top and right to the patient scores. al. Unfortunately. The weights of the second component have a very interesting pattern..4. almost all of the ﬁrst 27 weights are positive and the last 11 weights are negative.1:2] to print the weights to the screen it can be observed that those that correspond to the ﬁrst component are positive. et al. Namely.order(pca$scores[. A useful manner to plot both genes (cases) and patients (variables) is the biplot. center = TRUE. PRINCIPAL COMPONENTS ANALYSIS 139 This implies that large expression values on gene i co-vary positively with large deviations of gene j. Example 3.13 and 0. cor=TRUE.17).8) The resulting plot is given by Figure 7.2] Many of these genes are related to leukemia (Golub. 1985). scores=TRUE) o <.14. The positivity of the correlations also implies that the weights of the ﬁrst eigenvector have the same sign.7. we illustrate how it can be combined with principal components analysis.pc. 1999). which is in line with ﬁndings of Golub et al.gnames[o[3041:3051].5. (1999). By using -eigen(cor(golub))$vec[. this is not automatic in R so that caution is in order with respect to interpretation of the components. Biplot. Obviously the genes with the largest expression values from the ﬁrst component can be printed.2] golub. (1999). Here.cor=TRUE).biplot=TRUE. which .9999). however. The ﬁrst and the last ten gene names with respect to the values on the second component can be printed by the following. Thus the second component contrasts the ALL patients with the AML patients. so that these can be taken to be positive for all patients (Horn & Johnson.expand=0. concentrate on the second component because it appears to be more directly related to the research intentions of Golub et. We shall. which is based on a two-dimensional approximation of the data very similar to principal components analysis.2]) golub. Thus the ﬁrst component is almost equal to the sum of the variables (the correlation equals 0. > biplot(princomp(data.cex=0. > > > > pca <.

o2. we use a two-sample t-test. Example 4.cl) o1 <.]. and MCM3.as. ”Op”.c(o1. or ”MCM” in their names and collect the corresponding row numbers.frame(P[oo.o3) This yields 110 genes. Golub et al. CLUSTER ANALYSIS AND TREES are scaled to unit length by the speciﬁcation cor.data.golub.Z %*% -K$vec[.2]) o <. 1.eigen(cor(Z)) P <. In order to identify genes in directions of large variation we use the scores on the ﬁrst two principal components.12. data(golub.o[pt[o]<0.golub. scale = TRUE)) K <.gnames[. center = TRUE. function(x) t. In order to select those that do have an experimental eﬀect.value) This yields 34 genes.2]) o3 <.apply(golub.140 CHAPTER 7.names= oo) attach(leu) The scores on the ﬁrst two principal components of the selected genes are stored in the data frame leu. Z <.grep("MCM".matrix(scale(golub. package = "multtest") factor <. We select genes which carry ”CD”. cl <.method="single") plot(cl) .2]) o2 <.fac)$p.1:2] leu <.test(x ~ oo <.hclust(dist(leu. row.grep("CD". of which the row numbers are selected in the vector oo. (1999) mention that among genes which are useful for tumor class prediction there are genes that encode for proteins critical for S-phase cell cycle progression such as Cyclin D3. it seems that there are several sub-clusters of genes. pt <.factor(golub. The genes that belong to these clusters can be identiﬁed by hiearchical cluster analysis. Critical for S-phase.grep("Op".01] gol.gnames[.method="euclidian"). Op18. It can be seen that the patients are clearly divided in two groups corresponding to ALL and AML. From the plotted component scores in Figure 7.gnames[.golub.

CD95) The cluster with rows 504. 313. see Jolliﬀe (2002) for a complete treatment of the principal component analysis.2]. low affinity IIb. > a <.integer(rownames(leu)[cl$order]) > for (i in 1:length(a)) cat(a[i]. It can also be used to ﬁnd gene expressions which are highly dependent with a phenotypical variable. Principal components analysis can be useful in identifying clusters of genes in a lower dimensional space.gnames[a[i]. The correlation coeﬃcient measures the degree of dependency between pairs of gene expression values.12. some row numbers of genes are less readable because the points are very close. 1756. When these directions can be represented well by the ﬁrst two components a biplot helps to simultaneously visualize genes and patients. and 893 consists of antigenes.5 Overview and concluding remarks Single linkage cluster analysis can be applied to explore for groups in a set of gene expressions.7. 7. This illustrates that genes with similar functions may indeed be close with respect to their gene expression values. cerevisiae) 3 with row numbers 2289 and 2430 appear adjacent to each other.5.golub. Unfortunately. receptor for (CD32) 2874 GB DEF = Fas (Apo-1."\n") 1910 FCGR2B Fc fragment of IgG. 3 .as.13 various clusters of genes are apparent that also appear in Figure 7. OVERVIEW AND CONCLUDING REMARKS 141 From the tree (dendrogram) in Figure 7. The genes MCM3 Minichromosome maintenance deﬁcient (S. It is reassuring to ﬁnd in applications that the conﬁdence interval for a correlation coeﬃcient is small. 3 The ordered genes can be obtained from the object cl as follows. When groups are present a k-means cluster analysis can be applied in combination with the bootstrap to estimate conﬁdence intervals for the cluster means. Principal components analysis is very useful for ﬁnding directions in the data where the gene expression values vary maximally. (1999) data. Compare the results to those above. Recall that we did various analysis on the expression data of the CCND3 Cyclin D3 gene of the Golub (1999) data. (b) Remove the outlier. (c) Use k-means cluster analysis. (b) Produce of combined boxplot separately for the ALL and the AML expression values.142 CHAPTER 7.6 Exercises 1. CLUSTER ANALYSIS AND TREES 7. Do the conﬁdence intervals for the cluster means overlap? 2. You will have to modify the code here and there. (a) Plot the data and invent a manner to ﬁnd the row number of the outlier. Close to CCND3 Cyclin D3. In the example on MCM3 a plot shows that there is an outlier. (c) Perform the bootstrap to construct a conﬁdence interval. 4. Cluster analysis on part of Golub data. Are the two clusters according to the diagnosis of the patient groups? (d) Perform a bootstrap on the cluster means. (c) Compare the smallest distances with those among the Cyclin genes computed above. (a) Use genefilter to ﬁnd the ten closed genes to the expression values of CCND3 Cyclin D3. (b) Use single linkage cluster analysis to see whether the three indicates two diﬀerent groups. . (a) Produce a chatter plot of the gene expression values using showing diﬀerent symbols for the two groups. Cluster analysis on the ”Zyxin” expression values of the Golub et al. Give their probe as well as their biological names. Compare it with that on the basis of CCND3 Cyclin D3 and comment of the similarities. test the correlation coeﬃcient. MCM3. What is your conclusion? 3. 2.5  . (e) Plot the genes in a plot of the ﬁrst two principal components.5 1 0. 0.7.8 1 0. EXERCISES 143 (a) Select the oncogenes from the Golub data and plot the tree from a single linkage cluster analysis.8 1 −0.  −0. and 1.8.8  .     1 −0.5 −0. (b) How much variance represents the ﬁrst component corresponding to the second matrix? (c) Verify that the ﬁrst eigen vector of the second correlation matrix has identical signs. Are the ﬁrst three larger than one? (d) Program a bootstrap of the largest ﬁve eigenvalues.8 1 −0.001.5 1 0. (b) Do you observe meaningful clusters. 0. Report the dimensionality of the data matrix with gene expressions. Report the largest ﬁve.644529e-17. -7.5 −0.500000e+00. . Compute the corresponding ANOVA p-values of all gene expressions.2.  0. 5. Construct the expression set with the pvalues smaller than 0. 6.8 0. B2.500000e+00. Principal Components Analysis on part of the ALL data.5 −0. 0.8 1 −0.8 0. (d) select the receptor genes and answer the same questions. and B3.6. . (b) Are the correlations between the patients positive? (c) Compute the eigenvalues of the correlation matrix. Report the bootstrap 95% conﬁdence intervals and draw relevant conclusions.8 1 (a) Verify that the eigenvalues of the matrices are 1. Some correlation matrices.2. (c) Select the antigenes and answer the same questions. 1. 2.6. (a) Construct an expression set with the patients with B-cell in stage B1. 2 735 12 2 937 968 35 32 28 38 31 34 29 30 33 37 36 1066 2821 938 −2 1829 2920 2922 2921 1069 1901 2553 2734 2124 2656 1778 1413 378 −3 2664 2663 829 −3 −2 −1 0 1 2 Comp.0 24 1030 15 13 738 16 2489 4 26 19 21 1206 1 27 1334 2266 2939 1732 1882 1042 717 5 2386 839 1995 2020 345 2829 746 1037 523 866 8 2673 1109 394 7 3046 2851 1585 1598 22 703 422 2702 10811455 2645 1939 96 1909 801 462 31834 1271 2307 2289 329 1060 2794 563 2616 19592801 1640 9 1368 621 2418 2002 1978 2410 1817 2950 1086 2343 323 648 1653 2347 11 1920 2179 1916 23 2627 561 713 182725 1006 259 1524 922 1542 522 126 18 1869 1638 2889 202 704357376 23 134810451642 2466 2122 838 2593 25 1145 106 246 571 12451445 546 1856 2955 2356 984 2786 313 2265 494 244 1887 14 17 1110 0 1 0.5 0.0 −0.0 −0.5 .1 Figure 7.5 1652 2937 1556 786 1396 1754 896 2589 932 888 1448 2172 2749 2198 1774 566 108 1911 1665 803 766 808 2813 68 2600 −1 0. CLUSTER ANALYSIS AND TREES −1.5 2 20 1162 377 2459 Comp.144 CHAPTER 7.0 2065 0.14: Biplot of selected genes from the golub data. −1. Chapter 8 Classiﬁcation Methods In medical settings groups of patients are often diagnosed into classes corresponding to types of diseases. More generally. The speciﬁcity can be summarized in a single number by the area under the curve of a receiver operator curve. support vector machine and neural network are frequently applied to solve classiﬁcation problems. Two other methods to predict disease class from gene expression data are the support vector machine and the neural network. In bioinformatics the question arises whether the diagnosis of a patient can be predicted by gene expression values? Related is the question which genes play an important role in the prediction of class membership. This will be explained and illustrated. Many classiﬁcation methods have been developed for various scientiﬁc purposes. A similar question is the prediction of micro RNA’s from values of folding energy. In bioinformatics. A validation set will be used to evaluate the predictive accuracy. or microRNA’s it may be of importance to classify these on the basis of certain measurements. mRNA’s. for objects like proteins. 145 . To evaluate the quality of prediction the fundamental concepts of sensitivity and speciﬁcity are frequently used. It will brieﬂy be explained what these methods are about and how these can be applied. methods such as recursive partitioning. In this chapter you learn what recursive partitioning is and how to use it. 1 Per microRNA the 1001 energy values were arranged to have increasing order. 1981). CLASSIFICATION METHODS 8. Given a set of 3424 diﬀerent microRNA’s the minimum folding energy was computed for each of these. Zuker & Stiegler. as follows. This procedure yielded a total of 3424 p-values. similar as for empirical distributions in the previous chapter. This property can be used as a test to discriminate microRNA’s from non-microRNA’s (Bonnet. This yielded per microRNA 1000 diﬀerently shuﬄed sequences of nucleotides for which the minimum folding energy is computed.1: Frequencies empirical p-values lower than or test positive test negative p ≤ 0. Next. The same procedure is conducted for non-microRNA molecules which were taken as sequences with similar length and nucleotide percentages. One of these properties is that microRNA’s have the capacity to fold in a certain hairpin type of structure.01 p > 0.. for each microRNA the order of the nucleotides was shuﬄed with replacement 1000 times.1 Classiﬁcation of microRNA The subject of making a correct medical diagnosis is highly similar to that of correctly classifying microRNA. total 3424 3424 6848 I am obliged to Sven Warris for computing the minimum energy values. Classiﬁcation of Micro RNA. Table 8. 2004).01 is given in Table 8. If the minimum folding energie of the original microRNA is the smallest. Such a structure typically exhibits a small minimum folding energy (Zuker.01 microRNA 2973 451 non microRNA 33 3391 total 3006 3842 1 equal to 0. The number of sequences with p-values below the threshold value 0. then the empirical p-value is zero.146 CHAPTER 8. In order to identify microRNA’s from arbitrary sequences its characterizing properties are used to distinguish non-microRNA from microRNA molecules. MicroRNA are small RNA molecules with important functions in cell growth and disease development. .1.01. 2003. et al. Then the number of minimum folding energies below that of the original microRNA is counted and divided by 1001 as the p-value. Example 1. Predictive value positive = P V + = P (microRNA|test positive) = 2973 = 0.8826. ROC TYPES OF CURVES 147 From the frequency Table 8. The predictive value negative is the probability that the sequence is not a microRNA given that the test is negative. (1999) data that the expression values of gene CCND3 Cyclin D3 tend to be greater for ALL patients. and the predictive power can be computed in order to evaluate the quality of the test. From the estimated conditional probabilities it can be concluded that the test performs quite well in discriminating between microRNA’s from non-microRNA’s.9903. In particular.8.8682. 3842 Thus when the test is negative we are 88.1. In particular. for gene expression values larger than a cutoﬀ we declare the test “positive” in the sense of indicating ALL. the sensitivity. Thus sensitivity = P (true positive) = P (test positive|microRNA) = 2973 = 0.90% certain that the sequence is indeed a microRNA.9890 3006 Thus when the test is positive we are 98. Thus speciﬁcity = P (true negative) = P (test negative|no microRNA) = 3391 = 0. 3424 For practical applications of a test the predictive power is of crucial importance.26% certain that the sequence is not a microRNA. the predictive value positive is the probability that the sequence is a microRNA given that the test is positive. 8. The sensitivity is the probability that the test is positive given that the sequence is a microRNA (true positive). That is. 3424 The speciﬁcity is the probability that the test is negative given that the sequence is not a microRNA (true negative). the speciﬁcity.2 ROC types of curves In Chapter 2 we have observed with respect to the Golub et al.2. We may therefore use these as a test for predicting ALL using a certain cutoﬀ value. Predictive value negative = P V − = P (no microRNA|test negative) = 3391 = 0. By doing . true <. so that the true positive rate is 25/27=0. the cut oﬀ point 2.true gol. there are no expression values equal to inﬁnity. The procedure to draw the ROC curve starts with cutoﬀ point inﬁnity. To brieﬂy indicate the origin of the terminology.pred ALL not ALL ALL 25 1 notALL 2 10 There are 25 ALL patients with expression values greater than or equal to 1. > data(golub.93. see Table 8.77 is taken and values greater than or equal to 2. Example 1.gol.77 are tested as positive. package = "multtest") > gol. The receiver operator characteristic (ROC) is a curve where the false positive rates are depicted horizontally and the true positive rates vertically. This yields one true positive implying a More detailed information can be obtained from a wikipedia search using ”ROC curve”.09."FALSE").148 CHAPTER 8. The larger the area under the ROC curve. imagine that the test results are a characteristic received by an operator.2. (1999) in row 1042 of the matrix golub.levels=c("TRUE"."notALL")) > table(gol. CLASSIFICATION METHODS so the corresponding true and false positives can be computed for each cutoﬀ value. For this cutoﬀ value there is one false positive because one patient without ALL has a score larger than 1.pred <.27.27. labels=c("ALL". the better the test is because then low false positive rates go together with large true positive rates. so there is no patient tested positive.27.factor(golub[1042. (1999) data are sorted in decreasing order. Obviously.cl. The expression values for gene CCND3 Cyclin D3 from the Golub et al. Hence. Example 2. the false positive rate is 1/11 = 0.pred. 2 .]>1. Next.true) gol. For such a cutoﬀ point we can produce a table with TRUE/FALSE frequencies of predicting ALL/not ALL.factor(golub.27.labels= c(" ALL".levels=0:1."not ALL")) > gol.2 These ideas are illustrated by several examples. Now consider cutoﬀ point 1. For the sake of illustration we consider the prediction of ALL from the expression values for gene CCND3 Cyclin D3 from Golub et al. 8 1.1. see Figure 8. which implies a false positive rate of 1/11=0.09. However.4 0.81) in Figure 8.81.4 0.0 0.51. This process goes on (see Table 8.6 0.6 0.2. 0. there is one patient without ALL having expression value 1. To indicate this there is a vertical line drawn in the ROC curve from point (0.2) until the smallest data point -0. 1.81) to (0. Now consider the next cutoﬀ point 1. Figure 8. .4 0. 0. This is indicated by the end point (1.81) and the horizontal line from (0. There are 22 ALL patients with expression values greater than or equal to 1. whom receives therefore a positive test. 0. 0. so that the false positive rate is 11/11 and the true positive rate is 27/27.2 0.74 is taken as cutoﬀ point.0 0. There are 22 ALL patients with expression values greater than or equal to 1. so that the true positive rate is 22/27=0.0 0.81).8 1.09.2 0. 1) in the plot at the top on the right hand side.6 0.0 0.45. For this point all patients are tested positive.0 0. For this cutoﬀ value there are no false positives because all patients without ALL have expression values lower than 1. so that the true positive rate is again 22/27=0.52.4 0.1: ROC plot for expression values of CCND3 Cyclin D3.0 False positive rate False positive rate Figure 8.81.45.2. the false positive rate is 0 and the true positive rate is 0.45.8 True positive rate True positive rate 0. In the ROC curve this is indicated by point (0.8 1.2 0. see second row of Table 8. Hence.0 0.0 0. 0) to point (0. Hence. the number of false positives increases from zero to one. ROC TYPES OF CURVES 149 true positive rate of 1/27.6 0.1. Now consider cutoﬀ point 1.09.52.2 0.8.81. For this cutoﬀ value there are no negatives so that the false positive rate is zero.2: ROC plot for expression values of gene Gdf5. (1999) data the organisms are 38 patients which have measurements on 3051 genes.96. Hence.prediction(golub[1042. · · · .performance(pred.]. "fpr" ) plot(perf) It seems clear that the expression values are better in testing for ALL when the curve is very steep in the beginning and attains its maximum value soon. The ROC curve for the expression values of gene Gdf5 is given by Figure 8.true <. This corresponds to the area under the curve of 0.2. which is small. "tpr".true and use functions from the ROCR package. It can be observed that the true positive rate is much lower as one moves on the horizontal axis from the left to the right. library(ROCR) gol.labels= c("TRUE". The classes consist of diagnosis of patients into the ALL class (27 patients) and the AML class (11 patients).cl. 8. To do so we construct an appropriate factor with the value TRUE for ALL and FALSE for not ALL gol.levels=0:1.35. For instance. In such a case the true positive rate is large for a small false positive rate.factor(golub. In practical applications one is often interested in a single optimal cut-oﬀ value and in combining several predictors in a decision scheme."FALSE")) pred <. A manner to express the predictive accuracy of a test in a single number is by the area under the curve."auc") we obtain that the area under the curve is 0.1. Using the function performance(pred.150 CHAPTER 8.3 Classiﬁcation trees The purpose of classiﬁcation is to allocate organisms into classes on the basis of measurements on attributes.true) perf <. A tree model resembles that of a linear model. where the criterion is the factor indicating class membership and the predictor variables are the gene expression values. gol. CLASSIFICATION METHODS It is obviously helpful to use a computer for producing an ROC such as in Figure 8. the expression values of CCND3 Cyclin D3 are suitable for discrimination between ALL and not ALL (AML). x38 } . This illustrates that genes may express large diﬀerences with respect to prediction of the disease status of patients. (1999) data the gene expression values {x1 . which is large. In case of. for instance. the Golub et al. in case of the Golub et al. For instance. Venables. these can not be generalized to other samples so that these are of limited scientiﬁc value.. CLASSIFICATION TREES 151 can serve as predictors to form a decision tree. if xj < t. Example 1. 2000). the threshold value t on which the decision is based should be optimal given the predictor. 1984. Chambers & Hastie. Obviously. Optimal gene expressions. 3051 for instance.025 ALL2 0/10/0 ALL1 ALL2 AML AML 0/0/10 Figure 8. The rpart package automatically selects genes which are important for classiﬁcation and neglects others. Suppose microarray expression data are available with respect to patients suﬀering from three types of . genea< 0.4: Classiﬁcation tree for gene for three classes of leukemia. A further problem is that of overﬁtting where additional nodes of a tree are added to increase prediction accuracy.8. then we have a tremendous gene (variable) selection problem. Prevention of such overﬁtting is called pruning and is automatically done by the rpart function. When many predictor variables are involved.9371 4 3 ALL1 10/0/0 0 1 2 genea< 3. Many basic ideas are illustrated by an elementary example. then patient j is AML. & Ripley. 1997). which is implemented in the rpart package (Therneau & Atkinson. then patient j is ALL. A training set is used to estimate the threshold values that construct the tree. and otherwise if xj ≥ t. Such can be estimated by a regression tree (Breiman et al. 1992. When such nodes are speciﬁc for the training sample set.3: Boxplot of expression values of gene a for each leukemia class.3. Figure 8. 152 CHAPTER 8. CLASSIFICATION METHODS leukemia abbreviated as ALL1, ALL2, and AML. Gene A has expression values from the populations (patient groups) N (0, 0.52 ) for ALL1, N (2, 0.52 ) for ALL2, and N (4, 0.52 ) for AML. The script below generates thirty expression values for gene A, the patients of the three disease classes, and the estimates of the classiﬁcation tree. set.seed(123); n<-10 ; sigma <- 0.5 fac <- factor(c(rep(1,n),rep(2,n),rep(3,n))) levels(fac) <- c("ALL1","ALL2","AML") geneA <- c(rnorm(10,0,sigma),rnorm(10,2,sigma),rnorm(10,4,sigma)) dat <- data.frame(fac,geneA) library(rpart) rp <- rpart(fac ~ geneA, method="class",data=dat) plot(rp, branch=0,margin=0.1); text(rp, digits=3, use.n=TRUE) From the boxplot in Figure 8.3 it can be observed that there is no overlap of gene expressions between classes. This makes gene A an ideal predictor for separating patients into classes. By the construction of the gene expression values x1 , · · · , x30 we expect the following partition. If xi < 1, then ALL1, if xi is in interval [1, 3], then ALL2, and if xi > 3, then AML. From the estimated tree in Figure 8.4 it can be observed that the estimated splits are close to our expectations: If xi < 0.971, then ALL1, if xi is in [0.9371, 3.025], then ALL2, and if xi > 3.025, then AML. The tree consists of three leaves (nodes) and two splits. The prediction of patients into the three classes perfectly matches the true disease status. Obviously, such an ideal gene need not exist because the expression values overlap between the disease classes. In such a case more genes may be used to build the classiﬁcation tree. Example 2. Gene selection. Another situation is where Gene A discriminates between ALL and AML and Gene B between ALL1 patients and ALL2 or AML patients and Gene C does not discriminate at all. To simulate this setting we generate expression values for Gene A from N (0, 0.52 ) for both ALL1 and ALL2, and from N (2, 0.52 ) for AML patients. Next, we generate expression values for Gene B from N (0, 0.52 ) for ALL1 and from N (2, 0.52 ) for ALL2 and AML. Finally, we generate for Gene C from N (1, 0.52 ) for ALL1, ALL2, and AML. For this and for estimating the tree, we use the following script. 8.3. CLASSIFICATION TREES 153 set.seed(123) n<-10 ; sigma <- 0.5 fac <- factor(c(rep(1,n),rep(2,n),rep(3,n))) levels(fac) <- c("ALL1","ALL2","AML") geneA <- c(rnorm(20,0,sigma),rnorm(10,2,sigma)) geneB <- c(rnorm(10,0,sigma),rnorm(20,2,sigma)) geneC <- c(rnorm(30,1,sigma)) dat <- data.frame(fac,geneA,geneB,geneC) library(rpart) rp <- rpart(fac ~ geneA + geneB + geneC, method="class",data=dat) Note the addition in the model notation for the rpart function.3 It is convenient to collect the data in the form of a data frame.4 From the boxplot in Figure 8.5 it can be seen that Gene A discriminates well between ALL and AML, but not between ALL1 and ALL2. The expression values for Gene B discriminate well between ALL1 and ALL2, whereas those of Gene C do not discriminate at all. The latter can also be seen from the estimated tree in Figure 8.6, where Gene C plays no role at all. This illustrates that rpart automatically selects the genes (variables) which play a role in the classiﬁcation tree. Expression values on Gene A larger than 1.025 are predicted as AML and smaller ones as ALL. Expression values on Gene B smaller than 0.9074 are predicted as ALL1 and larger as ALL2. Hence, Gene A separates well within the ALL class. Example 3. Prediction by CCND3 Cyclin D3 gene expression values. From various visualizations and statistical testing in the previous chapters, it can be conjectured that CCND3 Cyclin D3 gene expression values form a suitable predictor for discriminating between ALL and AML patients. Note, however, from Figures 2.2 and 8.7 that there is some overlap between the expression values from the ALL and the AML patients, so that a perfect classiﬁcation is not possible. By the function rpart the regression partitioning can be computed as follows. > library(rpart);data(golub); library(multtest) > gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) > gol.rp <- rpart(gol.fac ~ golub[1042,] , method="class") 3 4 See Chapter 11 of the manual ”An Introduction to R” for more on model notation. See Chapter 6 of the manual ”An Introduction to R” for more on data frames. 154 CHAPTER 8. CLASSIFICATION METHODS genea< 1.025 2 1 geneb< 0.9074 AML 0/0/10 0 −1 ALL1 10/0/0 ALL1 ALL2 AML ALL2 0/10/0 Figure 8.5: Boxplot of expression values of gene a for each leukemia class. Figure 8.6: Classiﬁcation tree of expression values from gene A, B, and C for the classiﬁcation of ALL1, ALL2, and AML patients. > predictedclass <- predict(gol.rp, type="class") > table(predictedclass, gol.fac) gol.fac predictedclass ALL AML ALL 25 1 AML 2 10 Note that (25 + 10)/38 · 100% = 92.10% of the ALL/AML patients are correctly classiﬁed by gene CCND3 Cyclin D3. By the function predict(gol.rp,type="class") the predictions from the regression tree of the patients in the two classes can be obtained. The factor gol.fac contains the levels ALL and AML corresponding to the diagnosis to be predicted. The predictor variable consists of the expression values of gene CCND3 Cyclin D3. The output of recursive partitioning is assigned to an object called gol.rp, a list from which further information can be extracted by suitable functions. A summary can be obtained as follows. > summary(gol.rp) Call: 8.3. CLASSIFICATION TREES rpart(formula = gol.fac ~ golub[1042, ], method = "class") n= 38 CP nsplit rel error xerror xstd 1 0.7272727 0 1.0000000 1.0000000 0.2541521 2 0.0100000 1 0.2727273 0.5454545 0.2043460 155 Node number 1: 38 observations, complexity param=0.7272727 predicted class=ALL expected loss=0.2894737 class counts: 27 11 probabilities: 0.711 0.289 left son=2 (26 obs) right son=3 (12 obs) Primary splits: golub[1042, ] < 1.198515 to the right, improve=10.37517, (0 missing) Node number 2: 26 observations predicted class=ALL expected loss=0.03846154 class counts: 25 1 probabilities: 0.962 0.038 Node number 3: 12 observations predicted class=AML expected loss=0.1666667 class counts: 2 10 probabilities: 0.167 0.833 26 [1] 0.03846154 The expected loss in prediction accuracy of Node number 2 is 1/26 and that of Node number 3 is 2/12. This equals the probabilities from the class counts. The primary splits gives the estimated threshold value. To predict the class of the individual patients one may use the function predict, as follows. > predict(gol.rp,type="class") 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL ALL AML ALL ALL ALL 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 AML ALL ALL ALL ALL ALL ALL AML ALL AML AML AML AML AML AML AML AML AML Levels: ALL AML 156 CHAPTER 8. CLASSIFICATION METHODS Hence, Patient 17 and 21 are erroneously predicted as AML and Patient 29 is erroneously predicted in the ALL class. A more precise output is obtained by asking for the probability of class membership. > predict(gol.rp, type="prob") ALL AML 1 0.9615385 0.03846154 2 0.9615385 0.03846154 etc. Based on this the probability of patient 21 to have ALL is 0.16 and that to have AML is 0.83. −0.5 0.0 0.5 1.0 1.5 2.0 2.5 golub[1042, ]>=1.199 ALL 25/1 ALL AML AML 2/10 Figure 8.7: Boxplot of expression values from gene CCND3 Cyclin D3 for ALL and AML patients Figure 8.8: Classiﬁcation tree of expression values from gene CCND3 Cyclin D3 for classiﬁcation of ALL and AML patients. Example 4. Gene selection of the Golub (1999) data. By recursive partitioning it is possible to select among the genes of Golub et al. (1999) those which give the best partitioning. For the latter to work we have to specify the gene expressions as the variables (columns). For this we use the transposition operator t. To facilitate reading the output we add gene 1 to gene 3051 as column names. 8.3. CLASSIFICATION TREES 157 library(rpart);data(golub); library(multtest) row.names(golub)<- paste("gene", 1:3051, sep = "") goldata <- data.frame(t(golub[1:3051,])) gol.fac <- factor(golub.cl,levels=0:1, labels= c("ALL","AML")) gol.rp <- rpart(gol.fac~., data=goldata, method="class", cp=0.001) plot(gol.rp, branch=0,margin=0.1); text(gol.rp, digits=3, use.n=TRUE) golub.gnames[896,] Inspection of the plot yields gene ”FAH Fumarylacetoacetate” as the predictor by which the two classes of patients can be predicted perfectly. In order to further illustrate possibilities of classiﬁcation methods we use the ALL data collected by Chiaretti, et al. (2004), see also Chapter 6. Example 5. Application to the Chiaretti (2004) data. With respect to the ALL data we want to predict from the gene expressions the diagnosis of Bcell State B1, B2, and B3. Since the complete set of 12625 gene expressions is too large, we select the genes with diﬀerent means over the patients groups. It is obvious that only these gene can contribute to the prediction of the disease states. In particular we select the gene with ANOVA p-value is smaller than 0.000001. library("hgu95av2.db");library(ALL);data(ALL) ALLB123 <- ALL[,ALL$BT %in% c("B1","B2","B3")] pano <- apply(exprs(ALLB123), 1, function(x) anova(lm(x ~ ALLB123$BT))$Pr[1]) names <- featureNames(ALL)[pano<0.000001] symb <- mget(names, env = hgu95av2SYMBOL) ALLBTnames <- ALLB123[names, ] probedat <- as.matrix(exprs(ALLBTnames)) row.names(probedat)<-unlist(symb) The probe symbols are extracted from the hgu95av2SYMBOL environment and used as row names to facilitate readability of the resulting tree. There are 78 patients selected and 29 probes. The recursive partitioning to ﬁnd the tree can be performed by the following script. > diagnosed <- factor(ALLBTnames$BT) > tr <- rpart(factor(ALLBTnames$BT) ~ ., data = data.frame(t(probedat))) > plot(tr, branch=0,margin=0.1); text(tr, digits=3, use.n=TRUE)

05 0.026 0.85 0. then the patient is predicted to be in state (class) B1. type="class") predicted.895 0.026 0.026 0.00 B1 B1 04007 0.026 0. The resulting tree in Figure 8.11 0.05 0. diagnosis=factor(ALLBTnames$BT)) > print(out.895 0. The misclassiﬁcation rate is 10/78=0. but not zero.class diagnosis 01005 0.data. The matrix with frequencies of the predicted and true patient status is often called a “confusion table”.13 B2 B2 01010 0.1282051.13 B2 B2 08001 0.frame(predicted. then the predicted state is B2.895 0.90 B3 B3 08024 0.11 0. predicted. CLASSIFICATION METHODS > rpartpred <.85 0.13 B2 B2 08011 0.predict(tr.00 B1 B1 06002 0.026 0. and if it is larger than the predicted state it is B3. It may happen that the probability of the predicted class is close to that of the diagnosed.class.85 0.predict(tr.11 0.9 should be read as follows.13 B2 B2 04006 0.00 B1 B2 .probabilities.13 B2 B2 04008 0.13 B2 B3 08012 0.395.895 0.050 0. which is low.85 0.85 0. If gene expression MME is strictly smaller than the cutoﬀ value 8.probabilities <.00 B1 B1 04010 0.026 0.85 0.90 B3 B1 04016 0.predicted.13 B2 B3 08018 0.diagnosed) diagnosed rpartpred B1 B2 B3 B1 17 2 0 B2 1 33 5 B3 1 1 18 The rows to the left of the table give the frequencies of the predicted B cell stages and the columns on top the diagnosed B cell stages from the factor.class <.11 0. If the expression of LSM6 smaller than 4.digits=2) B1 B2 B3 predicted.predict(tr. An overview of the latter can be obtained as follows.026 0.158 CHAPTER 8. type="prob") out <. type="class") > table(rpartpred.192.85 0.050 0. B2 B3 159 For instance.. then highly similar ones are not selected anymore. Then a confusion matrix is constructed with the frequencies of true classes against predicted classes.05 in class B1. the misclassiﬁcation rate can be computed to evaluate the predictive accuracy. When such a future data set is not available.35 0.192 B2 1/33/5 B3 1/1/18 Figure 8.85 0. This can very well be seen . CLASSIFICATION TREES 09008 0.0 0. which is the diagnosed disease state. Note the reduction in variables from twenty nine to two in the actual construction of the tree. It can be instructive to leave out the variables selected from the data and to redo the analysis.45 0.3.8.026 0. Then the model is estimated from the training set and this is used to predict the class of the patients in the validation set. A generally applied manner to evaluate an estimated model is by its predictive accuracy with respect to a future data set.395 1389_at 38032_at 40440_at 307_at 37544_at 36711_at 34378_at 32977_at 32116_at 32716_at 36829_at 40729_s_at 37320_at 1173_g_at 40480_s_at 0.4 0.6 MeanDecreaseGini B1 17/2/0 LSM6< 4.9: rpart on ALL B-cel 123 data. In a construction like this the gene expressions (variables) are linearly dependent in the sense that once the ﬁrst gene is selected for the ﬁrst split.10: Variable importance plot on ALL B-cell 123 data. rf1 MME< 8.2 0. the sixth patient is with probability . Next.. Figure 8.90 in class B3 and with probability . it is common practice to split the available data in two parts: A training set and a validation set.65 MeanDecreaseAccuracy 1389_at 36711_at 38032_at 40440_at 40493_at 36829_at 34891_at 37544_at 37043_at 35769_at 34347_at 34333_at 307_at 32716_at 32977_at 0.55 0.13 . 05 and in the validation set is 7/39 = 0.predict(rpart.t..pred. In the setting of B-cell ALL data with State 1.setdiff(1:78.].18. The 78 patients in State 1.factor(ALLBTnames$BT[i])) rpart. and 3 the manner to split the data centers around randomly splitting the patients in two halves.rpart(Y ~ . CLASSIFICATION METHODS as a method to detect for over ﬁtting where the model estimates are so data speciﬁc that generalization to future data sets is in danger. 39.pred.est <.v B1 B2 B3 B1 6 1 0 B2 1 19 3 B3 1 2 6 The misclassiﬁcation rate in the training set is 2/39 = 0.t B1 B2 B3 B1 11 1 0 B2 0 12 0 B3 0 1 14 > rpart.t <. df[i.i) df <.factor(ALLBTnames$BT[noti])) rpart. Note that the diﬀerences mainly occur between State 2 and 3.].pred. Example 6. X =t(probedat)) rpart. Generally the prediction of disease state from the training set is better because the model is estimated from these data. as follows.160 CHAPTER 8. 2 or 3 can be split in two halves. This works like a classiﬁcation problem where the classes .pred. data = df. type="class") > table(rpart.4 Support Vector Machine A support vector machine ﬁnds separating lines (hyper planes) between groups of points. The same split of the data into training and validation set will be used for other methods as well.v.frame(Y = factor(ALLBTnames$BT). 2.data.predict(rpart. Training and validation. replace = FALSE) noti <.est. i <. 8.pred.sample(1:78.v <. type="class") > table(rpart.est.df[noti.pred. subset=i) rpart.

Note.8. B2. kernel = "linear") svmpred <. the excellent prediction properties are obtained by a very large number of estimated parameters. If such separating lines do exist in the data. 9. and 11. factor(ALLBTnames$BT)) svmpred B1 B2 B3 B1 19 0 0 B2 0 36 1 B3 0 0 22 The confusion matrix shows that the misclassiﬁcation rate of the three classes of B-cell ALL is 1/78=0. type = "C-classification". Support vector machines do not automatically select variables and are designed for continuous predictor variables. data=df. from summary(svmest) that the number of support vectors per class equals 20. and B3. Example 2.svm(X.0128 is very small. Y.data. respectively. We shall use the same split as in Example 6 of the previous section.t(probedat) svmest <. X =t(probedat)) Y <. Example 1. Hence. X. we shall conﬁne with illustrating applications to gene expression data. The parameters for the support vector machine can be determined by the function svm from the e1071 package. The model is estimated by the training set and then the class of the patients in the validation set is predicted. SUPPORT VECTOR MACHINE 161 of patients are to be predicted from gene expression values.X <. library(e1071) df <. probability=TRUE) > table(svmpred.frame(Y = factor(ALLBTnames$BT). . so that the prediction is almost perfect. for class B1. however.4. Since the mathematical details are beyond the current scope. These have values for all input variables (genes) as can be obtained from dim(svmest$SV) and the coeﬃcient vectors dim(svmest$coefs). Application to the Chiaretti (2004) data.predict(svmest. A generally applied manner to evaluate the predictive quality of an estimated model is by splitting the data into a training and a validation set. This is because the optimization method behind it is based on quadratic programming by iterative algorithms which ﬁnd the globally optimal solution with certainty.factor(ALLBTnames$BT). as follows. then a linear support vector machine will ﬁnd these. Training and validation. the parameter estimates from the training set are sample speciﬁc and do not generalize with the same accuracy to the validation set. Example 1. Yv) Yv svmpredv B1 B2 B3 B1 5 0 0 B2 1 19 4 B3 2 3 5 The predictions of the disease states of the patients from the training set perfectly match the diagnosed states.factor(ALLBTnames$BT).25 and are therefore less accurate. CLASSIFICATION METHODS Yt <. however. of the classes of the patients from the validation set have misclassiﬁcation rate 10/39=0. We conﬁne with illustrating the method by two examples.factor(ALLBTnames$BT)[i]. Application to the Chiaretti (2004) data. Xv <.t(probedat).162 > > > > > CHAPTER 8.predict(svmest. Yt) Yt svmpredt B1 B2 B3 B1 11 0 0 B2 0 14 0 B3 0 0 14 > svmpredv <. Xt.] svmest <. Hence.5 Neural Networks Neural networks are nonlinear models consisting of nonlinear hyperplanes around classes of objects given a set of prediction variables (Ripley. Yt. To avoid having to many variables we randomly select a subset of 20 genes. The models can be estimated by the function nnet from the package that goes under the same name. > Y <. 8. Xv. 1996).predict(svmest.]. type = "C-classification".svm(Xt. The predictions. probability=TRUE) > table(svmpredv.X[i. kernel = "linear") svmpredt <. probability=TRUE) table(svmpredt. Yv <. Xt <.t(probedat) .X[noti.factor(ALLBTnames$BT)[noti] X <.X <.

Ytrain=Y[i]) Ytrain prednnt B1 B2 B3 B1 11 0 0 B2 0 14 0 B3 0 0 14 > prednnv <.]. type = "class") > table(pred. decay = 0. type = "class") > table(prednnv.frame(Y = Y.. .5.t.. size = 5.t <.data = df. df[noti. maxit = 500.decay = 0.t. df[i.predict(nnest. Training and validation. NEURAL NETWORKS 163 > library(nnet) > df <. + MaxNWts = 5000) > pred <.nnet(Y ~ .predict(nnest. Yval= Y[noti]) Yval prednnv B1 B2 B3 B1 4 1 0 B2 4 17 4 B3 0 4 5 The predictions on the training set have misclassiﬁcation rate zero and that on the validation set 13/39=0.subset=i. + maxit=500) > prednnt <.01. > nnest.8. The results from cross validation on the neural networks are as follows.01.data. size = 5. Example 2. 20)]) > nnest <. X = X[. Y) # prints confusion ma Y pred B1 B2 B3 B1 19 0 0 B2 0 36 0 B3 0 0 23 The confusion matrix shows that zero out of 78 patients are mis-classiﬁed.33. data = df. sample(ncol(X).predict(nnest.]. type = "class") > table(prednnt.nnet(Y ~ .

Recall from Chapter 3 that for a binomially distributed variable with yi successes out of ni it holds that the probability of yi successes out of ni equals P (Yi = yi ) = ni ! pyi (1 − pi )ni −yi . (1999) data we may model Yi = 1 if the patient is diagnosed as ALL and Yi = 0 if (s)he is diagnosed as AML.df.1). We use the CCND3 Cyclin D3 gene expression values as predictor.5. CCND3 Cyclin D3.164 CHAPTER 8. To will be convenient to compute the response by -golub.6 Generalized Linear Models Within the framework of generalized linear models the diagnosis of a patient is seen as a response. Example 1. yi !(ni − yi )! i for k = 0. the usefulness of generalized linear models will be illustrated with two examples.lower=FALSE) 5 One may also conveniently use a factor as response variable . The value of pi is closely related to one or more predictor variables x1 and x2 via a linear combination.439953*x)) pchisq(deviance(logitmod).]. ηi + 1 e 1 + exp(β0 + β1 xi1 + β2 xi2 ) Rather than going deeper into the details. xlim=c(-2.ilogit(-4.cl + 1.1) lines(x.df. In the Golub et al.].residual(logitmod).lower=FALSE) plot((-golub. xlab="CCND3 expression values ".seq(-2.residual(logitmod). CLASSIFICATION METHODS 8. · · · .5). family=binomial(link = "logit")) pchisq(deviance(logitmod). the linear model holds such that ηi = β0 + β1 xi1 + β2 xi2 . That is.. ylim = c(0. In case the response has the values healthy or disease for which it may be assumed that the binomial distribution holds with a succes probability pi . library(faraway) logitmod <.cl + 1) ~ golub[1042.cl + 1) ~ golub[1042. This yield 1 for ALL and 0 for not ALL5 . ylab="Probability of ALL") x <.844124 + 4. ni .glm((-golub. The predictors are linked to the succes probability via the so-called logit link pi = exp(β0 + β1 xi1 + β2 xi2 ) eηi = .

]. Here we use the gene expressions with greatest importance from the classiﬁcation tree in Section 8. From the summary of the output it can be seen that the estimated intercept is -4. > pred <.cl + 1) ~ golub[1042.fac) gol. The model ﬁts the data well. family = binomial(link = "logit")) Coefficients: Estimate Std.type="response") > 0. GENERALIZED LINEAR MODELS > summary(logitmod) 165 Call: glm(formula = (-golub.99. Application to the Chiaretti (2004) data.440 1.488 2."not ALL")) > table(pred.00284 ** Null deviance: 45.db").library(ALL).270 AIC: 22.849 -2. With respect to the ALL data we want model the diagnosis of B-cell State B1.library("hgu95av2. and B3 as a response.844 and the estimated slope is 4.gol. We assign the biological name to the predictor variables and estimate the generalized linear model.predict(logitmod.844 1. B2.levels=c(TRUE.data(ALL) . The factor representing these levels can be used as input for the response. Example 2.6.FALSE).labels=c("ALL".440. Both coeﬃcients are signiﬁcantly diﬀerent from zero. ] 4.984 0.fac pred.270 on 37 on 36 degrees of freedom degrees of freedom From Figure 8.fac <.00880 ** golub[1042.728 Residual deviance: 18.fac.11 it can be seen that the logit curve ﬁts the data fairly well. The goodness-of-ﬁt value of the model is computed from the chi-square distribution and equals .5 > pred.620 0.3. Error z value Pr(>|z|) (Intercept) -4. The predictive accuracy of the model may be obtained as follows.factor(pred. library(nnet).fac ALL AML ALL 26 2 not ALL 1 9 The diagnosis of the majority of patients is predicted correctly.8.

fac) fac predmn B1 B2 B3 B1 17 2 1 B2 1 31 5 B3 1 3 17 The model predict the diagnosed classes quite well.c("1389_at". env = hgu95av2SYMBOL)) fac <.367058 1.166 CHAPTER 8. Generalized linear models are statistical models which have to be estimated by an iterative process which may need some computation time.t(probedat)) mnmod <. family = binomial(link = "logit")) Coefficients: (Intercept) MME LSM6 SERBP1 B2 14.data.716513 2. It .multinom(fac ~ ."B3")] probedat <.predict(mnmod.names.655420 Std.14002 -0. CLASSIFICATION METHODS probe.222486 B3 17.ALL$BT %in% c("B1".ALL[.88298 Apart from the intercepts. Errors: (Intercept) MME LSM6 SERBP1 B2 16. the estimated coeﬃcients are signiﬁcantly diﬀerent from zero.."40440_at") ALLB123 <.names(probedat) <."35991_at"."B2".88298 AIC: 75.names.94908 4.] row. data = dat. > predmn <.unlist(mget(probe."B3")) dat <.exprs(ALLB123)[probe. family=binomial(link = "logit").104337 B3 -12.factor(ALLB123$BT.frame(fac.36158 4.data=dat) > summary(mnmod) Call: multinom(formula = fac ~ .313700 Residual Deviance: 59.8494635 -5.97896 1..424526 1.7415802 -5."B2".levels=c("B1".90584 4.names <.744425 2.type="class") > table(predmn.36959 1.

In addition. using statistical model building procedures may not designed to handle large amounts of predictor variables. Recursive partitioning to estimate a classiﬁcation tree performs very well on variable selection and pruning in order to discover as few variables (gene expressions) as possible for maximum predictive accuracy. On the other hand. but less well on validation sets. Even from a more pragmatic point of view such need not be devastating if the predictive accuracy is excellent.8 6 Exercises 1.7 Overview and concluding remarks Central themes in prediction methods are the face validity (clarity) of the model. Note that several methods have diﬀerent misclassiﬁcation rates with respect to the whole sample. be clear that when there are non-linear relationships between predictor variables and classes. the researcher does need to have some idea on which gene expressions (s)he want to use as predictors.g. better models than those estimated in the above example may certainly exist. the CART package (Breiman et al. and predictive accuracy on a validation set. Hence. Indeed. 1984) for further types of recursive trees. see e. Use recursive partitioning in rpart Some people may want to use the ade4TkGUI() . It is. the size of the model. Classiﬁcation tree of Golub data. however. but comparable rates on the validation sets. For many researchers it is of crucial importance to have a clear idea on what a method is essentially doing.. OVERVIEW AND CONCLUDING REMARKS 167 has the advantage that conﬁdence intervals or the signiﬁcance of the parameters can be estimated. support vector machines and neural networks typically use a large number of parameters to predict well on a test set. 8. However. furthermore. then nonlinear models should outperform linear ones6 .8.7. questionable whether a zero misclassiﬁcation rate is rational since patients may be misclassiﬁed by the diagnosis or very close to transferring from one state to the other. 8. It should. Some models and their estimation procedures are mathematically intricate and seem to be recollected in the mind of many researchers as black boxes. it seems obvious that classiﬁcation trees have great clarity.

Use the ﬁrst four letters of the alphabet for the column names. . we perform a small simulation study. Give explanations of the results. (b) Use rpart to construct a recursive tree and report the misclassiﬁcation rate.2. remain ill). Comment on the results. (c) Do the same for support vector machines. (c) Use rpart to construct the classiﬁcation tree with the genes that you found. CLASSIFICATION METHODS (a) Find a manner to identify an optimal gene with respect the Golub data to prediction of the ALL AML patients. (b) Explain what the code does. (e) Think through your results and comment on these. (d) Do the same for neural networks. For the ALL data from its ALL library the patients are checked for achieving remission. which is supposed not to have any relationship with leukemia. (a) Produce a sensitivity versus speciﬁcity plot for the gene expression values of CCND3 Cyclin D3. To obtain an idea on the misclassiﬁcation rate when there is no relation between the predictors and the factor indicating groups. The variable ALLCR has values CR (became healthy) and REF (did not respond to therapy. 3. Estimate a classiﬁcation tree and report the probability of misclassiﬁcation. Sensitivity versus speciﬁcity. Prediction of achieved remission.168 CHAPTER 8. 4. (c) Compute the area under the curve for sensitivity versus speciﬁcity curve. Comparing Classiﬁcation Methods. (b) In what sense does it resemble Figure 8. Does it have perfect predictions? (d) Find the row number of gene Gdf5. 2. (a) Construct a factor with 100 values one and two and a matrix with predictor variables of 500 by 4 with values from the normal distribution. Is it much worse? ."ecclass") (a) Use ecclass to construct a factor containing the ”cp”. (1999) data.8."gvh". Report the code and the missclassiﬁcation rate. Report the miss-classiﬁcation rate.”aac”.and ”pp”. A strategy of selecting genes is to compute the auc for each gene and to use the best 10 for further investigation. "aac".c("SequenceName"."alm2". (d) Predict the class by the tree.”gvh”. (e) Leaf out the upper variable in the classiﬁcation tree and re-estimate the tree.".”alm1”.read.fr/~torre/Recherche/Datasets/ downloads/ecoli/ecoli. Collect these in a vector and select the ten best.header = TRUE) colnames(ecoli) <. Report the misclassiﬁcation rate and the names of the genes that play a role in the tree.8. 5."lip". Gene selection by area under the curve.sep=".grappa."mcg".) ecoli <.table( "http://www.data". Hint: Use the addition notation."chg".”alm2”. Classiﬁcation Tree for Ecoli.”im”. Give the code. EXERCISES 169 (a) Construct an expression set containing the patients with values on the phenotypical variable remission and the gene expressions with a signiﬁcant p-value on the t-test with the patient groups CR or REF.univ-lille3. Compute the auc for each row with gene expressions of the Golub at al. The ecoli data can be download by the following: (Hint: Copy two separated lines into one before running it. (b) Use recursive partitioning to predict the remission.”lip”. (b) Construct a classiﬁcation tree using the variables ”mcg”. (c) Plot the tree and report the variables that play a role in the constructed tree."alm1". Is ”CCND3 Cyclin D3” among these? 6. 43 9 0. true positive rate.83 5 0.11 0.00 1.78 1.77 2.93 0.00 0.28 1 1.00 0.49 0. 1 indicates AML.11 0.13 10 −0.37 1.00 0.00 1.36 0.64 7 0. index 2 indicates ALL.59 2.81 0.09 0.02 0.96 0.170 CHAPTER 8.59 2.00 0.46 1. fp 0 0 0 0 fpr 0.74 0.28 1.33 1.46 8 0.73 0.07 0.82 0.74 index 2 2 2 2 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 cutoﬀ Inf 2.74 11 .96 0.13 −0.00 tp 0 1 2 3 21 22 22 23 24 25 25 26 26 26 26 26 26 26 27 27 27 27 tpr 0.64 0. .93 0.00 1.78 0.18 0.02 3 0.52 0 1. number of false positives. cutoﬀ points.85 0.77 2.12 2 1.09 0.64 0.83 0.96 0.89 0.12 1.00 0.09 0.96 0.27 0.78 0 1.73 0. data 1 2 3 4 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 2. .55 0. false positive rate.52 1.96 0.04 0.74 6 0.09 0.96 0.45 1.45 1.2: Ordered expression values of gene CCND3 Cyclin D3.45 1 1.11 2 1.91 1. number of true positives.00 0.49 8 0.45 0.96 1.45 .00 0.33 1 1.81 0. CLASSIFICATION METHODS Table 8.43 0.00 1.89 0.37 1 1.89 4 0.18 0. 0 −1 0 1 2 3 4 5 CCND3 expression values Figure 8. .2 0.11: Logit ﬁt to the CCND3 Cyclin D3 expression values.8 1.4 0.0 −2 0.8.6 0. EXERCISES 171 Probability of ALL 0.8. CLASSIFICATION METHODS .172 CHAPTER 8. It will be explained and illustrated how optimal pairwise alignment can be obtained. It will be explained and illustrated how such quantities can be computed.1 Using a query language It will be illustrated how the query language from the seqinr package can be used for various types of searches. However. > library(seqinr) > choosebank() [1] "genbank" [6] "refseq" [11] "hovergen" [16] "hoverclnu" "embl" "nrsub" "hogenom" "hoverclpr" "emblwgs" "hobacnucl" "hogenomdna" "homolens" 173 "swissprot" "hobacprot" "hogennucl" "homolensdna" "ensembl" "hovergendna" "hogenprot" "greview" . however. 9. to match patterns. it is of importance to compute quantities for DNA sequences such as the CG fraction. The idea is that highly similar sequences may have identical biological functions. to translate RNA into protein sequences. We will start. For expressing the similarity of sequences it is necessary to compute ﬁrst their optimal alignment. or.Chapter 9 Analyzing Sequences For many purposes in bioinformatics nucleotide or amino acid sequences are analyzed. Furthermore. for amino acid sequences. before we download anything. with a query language in order to download sequences. it is important to know which banks can be chosen. In this chapter you learn how to query online data bases. and to program pairwise alignments. the isoelectric point or the hydropathy score. "k=ccnd". Example 1.2 Getting information on downloaded sequences After sequences are downloaded in binary format it is essential to obtain information with respect to their accession number. et al. for answering questions about sequences from online data bases (Gouy. 9. > query("ccnd3hs". For this we shall temporary use the option virtual=TRUE to save time by preventing actual downloading.virtual=TRUE)nelem [1] 9 For many other combinations of search options we refer to the manual of the seqinr package and for a book length treatment with many examples to Charif et al."sp=homo sapiens AND k=ccnd3".virtual=TRUE)$nelem [1] 147 More speciﬁc: How many sequences ccnd sequences has genbank for the species homo sapiens. > choosebank("genbank") > query("ccnd3hs". Let’s download sequences related to the species homo sapiens and a gene name like ”CCND3”.174 CHAPTER 9. 1984). translation to amino acids.g. (2008). and annotation. . length. ANALYZING SEQUENCES "HAMAPnucl" "taxobacgen" "HAMAPprot" "hoppsigen" [21] "polymorphix" "emglib" [26] "nurebnucl" "nurebprot" There are many possibilities to use the query language e. How to do this will brieﬂy be illustrated by an example. actual elements."sp=homo sapiens AND k=ccnd3@") > ccnd3hs$nelem [1] 9 1 The results below are obviously time dependent.1 We may ask: How many ccnd sequences has genbank? > choosebank("genbank") > query("ccnd". We give a few examples to illustrate some of its possibilities.

5306. getLength) [1] "879" "879" "729" "211627" "879" "879" "879" "537" "559" "879" Let’s obtain the ﬁrst sequence and print its ﬁrst ﬁfteen nucleotides to the screen. .CCND3" "AL161651" "HUMCCND3PS... getName) [1] "AF517525.PE1" The length of the sequences can be obtained by the getLength function.2.6005.. getLength.CCND3" [5] "BC011616.1248. Some of these are getName. GETTING INFORMATION ON DOWNLOADED SEQUENCES 175 The sequences are downloaded in binary format..6760)" [3] " /gene=\"CCND3\"" [4] " /codon_start=1" [5] " /product=\"cyclin D3\"" [6] " /protein_id=\"AAM51826.PE5" "HUMCCND3A.1\"" [7] " /db_xref=\"GI:21397158\"" [8] " /translation=\"MELLCCEGTRHAPRAGPDPRLLGDQRVLQSLLRLEERYVPRASY" 2 Use double brackets to extract a sequence from a list.5465..CCND3" "AL160163.9. > sapply(ccnd3hs$req.CCND3" "HUMCYCD3A.CCND3" "AL160163. The symbol @ acts as a wildcard for any zero or other characters. and getAnnot.2115.CCND3" "CR542246" [9] "HUMCCNDB04.6141." [2] " 6593. To use these on a list containing sets of sequences the functionality sapply is very convenient. 2 > getSequence(ccnd3hs$req[[1]])[1:15] [1] "a" "t" "g" "g" "a" "g" "c" "t" "g" "c" "t" "g" "t" "g" "t" Its translation into amino acids can be obtained > getTrans(ccnd3hs$req[[1]])[1:15] [1] "M" "E" "L" "L" "C" "C" "E" "G" "T" "R" "H" "A" "P" "R" "A" as well as its annotation from the corresponding web page: > getAnnot(ccnd3hs$req[[1]]) [1] " CDS join(1051. getSequence. > sapply(ccnd3hs$req. getTrans. There are a number of useful functions available to obtain further information.2330. This is illustrated by extracting the NCBI accession numbers. 3 Computations on sequences A basic quantity to compute are the nucleotide and the dinucleotide frequencies. > GC(getSequence(ccnd3hs$req[[1]])) [1] 0. Example 1. To compute the frequencies we may extract the sequence from a list in order to use the basic function table. changing 2 into 3 makes it possible to count trinucleotides. Example 2. We are often interested in the fraction G plus C in general (GC). Indeed.6313993 .2) aa 25 ac 44 ag 64 at 29 ca 68 cc 97 cg 45 ct 78 ga gc 52 104 gg 76 gt 34 ta 16 tc 43 tg 82 tt 21 This will be quite useful in the next chapter.CCND3”. or starting from the ﬁrst position of the codon bases (GC1). G + C percentage. as follows. or third (GC3). > table(getSequence(ccnd3hs$req[[1]])) a c g t 162 288 267 162 This table can also be computed by the seqinr function count. which is more general in the sense that frequencies of dinucleotides can be computed. We shall continue with the ﬁrst result from the CCND3 (Cyclin D3) search with accession number ”AF517525. ANALYZING SEQUENCES FQCVQREIKPHMRKMLAYWMLEVCEEQRCEEEVFPLAMNYLDRYLSCVPTRKAQLQLL" GAVCMLLASKLRETTPLTIEKLCIYTDHAVSPRQLRDWEVLVLGKLKWDLAAVIAHDF" LAFILHRLSLPRDRQALVKKHAQTFLALCATDYTFAMYPPSMIATGSIGAAVQGLGAC" SMSGDELTELLAGITGTEVDCLRACQEQIEAALRESLREAAQTSSSPAPKAPRGSSSQ" GPSQTSTPTDVTAIHL\"" 9. > count(getSequence(ccnd3hs$req[[1]]). Frequencies of (di)nucleotides. the second (GC2).176 [9] [10] [11] [12] [13] " " " " " CHAPTER 9.

fx .84 0.30 0.83 1.94 0. 2006).06 1. The coeﬃcient rho and the corresponding z-scores will be computed from the sequence with NCBI accession number ”AF517525.19 0.81 1.length(ccnd3[[1]]) for (i in 1:(n .2) . say. It is also possible to compute the G + C fraction in a window of length 50 nt.50)) GCperc[i] <plot(GCperc. which is deﬁned as ρ(xy) = fxy . fx · fy where fxy ..70 > round(zscore(getSequence(ccnd3hs$req[[1]]). Example 3. the G + C percentage is largest when started at position three. GCperc <.78157 177 Hence.1 it can be seen that the G + C fraction changes drastically along a window of 50 nucleotides. The z-score is computed by subtracting the mean and dividing by the standard deviation (Palmeira.modele=’base’). respectively. x.47 1. With respect to over or under representation of dinucleotides there is a function ρ (rho) available. et al.type="l") GC(ccnd3[[1]][i:(i+50)]) By double() we ﬁrst create a vector. Rho and z-scores.9. COMPUTATIONS ON SEQUENCES > GC1(getSequence(ccnd3hs$req[[1]])) [1] 0. and fy are the frequencies of the (di)nucleotide xy. > round(rho(getSequence(ccnd3hs$req[[1]])).54 0.67 0. and y. From Figure 9. and to plot it along the sequence.CCND3”.28 1.6484642 > GC2(getSequence(ccnd3hs$req[[1]])) [1] 0.97 1.double() n <.51 1.3.69 0.2) aa ac ag at ca cc cg ct ga gc gg gt ta tc tg tt 0.03 0.4641638 > GC3(getSequence(ccnd3hs$req[[1]])) [1] 0. The latter is somewhat more sensitive for over and under representation. 8 0.87 -3.22 -1.60 -0. it may be interesting to construct a .64 ga 0.1: G + C fraction of sequence ”AF517525.08 -1.81 -0.54 gc gg gt ta 2.78 cc cg 0. ANALYZING SEQUENCES GCperc 0.98 ca 2. aa ac ag at -1.CCND3” along a window of length 50 nt.5 0 0.7 0. In case we have an amino acid sequence it may be useful to obtain a plot of the amino acid frequencies.67 2. but its z-score certainly is.10 The rho value for CG is not extreme.42 -6.86 6.18 tc tg tt -1.9 200 400 Index 600 800 Figure 9.63 ct 4. When we have translated the nucleotide sequence into an amino acid sequence.80 -2.6 0.178 CHAPTER 9. table(getTrans(ccnd3hs$req[[1]])) taborder <. Example 4.CCND3. Leu Ala Arg Glu Ser Thr Pro Gln Val Gly Asp Cys Lys Ile Met Tyr His Phe Trp Asn Stp 0 10 20 30 40 Leu Ala Arg Glu Ser Thr Pro Gln Val Gly Asp Cys Lys Ile Met Tyr His Phe Trp Asn Stp 0 10 20 30 40 Stop and amino−acid−counts Stop and amino−acid−counts Figure 9. produce a dotchart with amino acid frequencies. Comparing Amino acid frequencies.3: Frequency plot of amino acids from accession number AL160163. .aaa(names(taborder)) dotchart(taborder. and. Such can be useful for a ﬁrst impression on sequence similarity. translate and order it. next.lty=2) The script was run on both sequences AF517525. tab <.CCND3 resulting in Figure 9. We continue with the ﬁrst result from the CCND3 (Cyclin D3) search.3.2: Frequency plot of amino acids from accession number AF517525.9. The two sequences are highly similar with respect to amino acid frequencies. For amino acid sequences it may be of importance to compute the theoretical isoelectric point or the molecular weight of the corresponding protein.CCND3.pch=19.2 and 9. Figure 9.xlab="Stop and amino-acid-counts") abline(v=1. respectively. COMPUTATIONS ON SEQUENCES 179 plot expressing their frequencies.3.tab[order(tab)] names(taborder) <.CCND3 and AL160163.

getTrans) data(EXP) names(EXP$KD) <. Example 6. function(x) translate(s2c(x))) kdc <. coef) { #data are sequences f <. The unique names are lexicographically ordered and stored in the object kdc. α20 are available as KD data from the EXP list of the seqinr package.function(x) { freq <. getSequence) ccnd3transl <.180 CHAPTER 9. > pmw(getTrans(getSequence(ccnd3hs$req[[1]]))) [1] 32503. but smaller than one.sapply(ccnd3hs$req.NULL . The coeﬃcients α1 . which is deﬁned as a weighted sum 20 αi fi of amino acid coi=1 eﬃcients αi and the relative frequencies fi . ccnd3 <. which is the pH at which the protein has a neutral charge (Gasteiger. 2005).-kdc[order(names(kdc))] linform <.table(factor(x. levels = names(coef)))/length(x) return(coef %*% freq) } res <.EXP$KD[unique(names(EXP$KD))] kdc <. A function is deﬁned to compute the hydropathy score for a set of amino acid sequences. et al. The function computePI computes the theoretical isoelectric point of a protein.38 Note that it is easy to compute these for all downloaded proteins and to compare these.657579 The protein molecular weight can be computed as follows. · · · . Another important quantity is hydropathy score (Kyte & Doolittle.function(data.sapply(words().sapply(data. Hydropathy score. An example will illustrate how it can be computed. f) names(res) <.sapply(ccnd3. 1982) of proteins. > computePI(getTrans(ccnd3hs$req[[1]])) [1] 6. The scale is changed by the minus sign below so that hydrophilic proteins are positive. ANALYZING SEQUENCES Example 5. Isoelectric point.

sapply(ccnd3hsreq.0962 0.. kdc) > print(kdath. ccnd3nr1. Example 1. mismatch = 0) Views on a 879-letter BString subject . Roberts.digits=3) [1] 0.2659 0. the largest score is still much smaller than one.9. In the sequence with NCBI accession number ”AF517525. we seek the pattern ”cccggg” with zero mismatches as well as those with a single mismatch.linform(ccnd3transl.0874 0.g.. > subseq <. There are many relevant examples such as seeking for one of the stop codons UAG. We sustain with a brief example. The data set aaindex of the seqinr library contains more than ﬁve hundred sets of coeﬃcients for computing speciﬁc quantities with respect to proteins. 9.0874 0.4. et al.. ccnd3nr1."cccggg" > countPattern(subseq. eventually allowing for a speciﬁed number of mismatches. By the function c2s a sequence of characters is converted into a single string.2220 Indeed. library(seqinr) choosebank("genbank") query("ccnd3hs". MATCHING PATTERNS return(res) 181 } kdath <.0189 0. 2007).CCND3”. mismatch = 0) [1] 2 > matchPattern(subseq. getSequence) ccnd3nr1 <. so the conclusion is that there are no hydrophilic proteins among our sequences.c2s(ccnd3[[1]]) > ccnd3nr1 [1] "atggagctgctgtgttgcgaaggcacccggcacgcgccccgggccgggccggacccgcgg". UGA UAA in RNA.0874 0.4 Matching patterns A manner to investigate a long sequence is to search for identical patterns. Pattern match.0962 0."sp=homo sapiens AND k=ccnd3@") ccnd3 <. or recognition sequences of enzymes (e.1496 0. actcctacagatgtcacag Views: start end width [1] 26 31 6 [cccggc] [2] 37 42 6 [ccccgg] [3] 38 43 6 [cccggg] [4] 43 48 6 [gccggg] [5] 54 59 6 [cccgcg] [6] 119 124 6 [cccgcg] [7] 236 241 6 [ccctgg] [8] 303 308 6 [cctggg] [9] 512 517 6 [cccgtg] [10] 612 617 6 [cacggg] [11] 642 647 6 [cctggg] [12] 661 666 6 [tccggg] [13] 662 667 6 [ccgggg] [14] 808 813 6 [ccccgg] [15] 809 814 6 [cccggg] [16] 810 815 6 [ccgggg] The number of counted patterns allowing two mismatches is much larger.actcctacagatgtcacag Views: start end width [1] 38 43 6 [cccggg] [2] 809 814 6 [cccggg] > matchPattern(subseq. 9. . To ﬁnd this out these are aligned in a certain manner after which a similarity score can be computed. ccnd3nr1..182 CHAPTER 9. ANALYZING SEQUENCES Subject: atggagctgctgtgttgcgaaggcacccggcacg.. mismatch = 1) Views on a 879-letter BString subject Subject: atggagctgctgtgttgcgaaggcacccggcacg...5 Pairwise alignments Among the basic questions about genes or proteins is to what extent a pair of sequences are similar. In order to understand sequence alignment it is fundamental to have some idea about recursion. Then the alignment consists of a match. x3 = 8. Then we obtain x1 = 1.5. j).x[1]<-1 > for (i in 2:10) {x[i]<. respectively. The idea of recursion is to generate a sequence by deﬁning the current value as a function of the previous. mismatch. To do so it is convenient to start with building the (mis)match score matrix s(i. with x1 = 1. where GAT T A the minus sign indicates a gap. For example. etc. Another manner to deﬁne a sequence is by multiplying the previous value by a constant. consider the alignment G A − T T C . Note that for . 3. mismatch. as follows. match. PAIRWISE ALIGNMENTS 183 Example 1. Also we see that in fact xn = 2n . x2 = 2. gap. mismatch. j)th element s(i. x2 = 2. Its (i. A gap at a certain position of the sequences should be punished T by subtracting a score by d = 2. al. but still we do not know whether this alignment is optimal. In order to ascertain that the alignment is optimal we have to build an alignment score matrix F (i. j). Suppose we want to compute an alignment score for two small DNA sequences GAATTC and GATTA (Durbin et. j) has the value 2 in case of a match and the value -1 is case of a mismatch. > x<-double(). · · ·. 1998.2*x[i-1]-10} > x[10] [1] -4598 This illustrates basic ideas about recursively deﬁned sequences. Suppose that the ﬁrst element is one. Then the values of the sequence are x1 = 1. let xi = 2xi−1 with x1 = 1. and that the sequence is deﬁned by xi = xi−1 + 1. Another example would be xi = 2xi−1 − 10. 2. In order to compute the value x10 we may use R. We agree that a match between two letters should have the score +2 and a mismatch the score -1. Indeed. so that the score is 2+2−2+2+2−1 = 5. GA T T A match.18). match.9. gap. Then we have a match. x3 = 4. etc. this is as fundamental as counting. so that the score is 2 + 2 − 1 + 2 − 2 − 1 = 2. so that a value of the sequence can be computed without actually computing all previous elements. so that the sequence becomes 1. x3 = 3. Basic recursion. match. Now the question is whether this alignment is optimal in the sense that the score is maximal? The answer is: No! To see A this. p.. A possible alignment is G A A T − C . This is better. match. x1 = 1. respectively. j) = F (i. colnames(s) <. 0) = 0 and due to the gap penalties we take F (i. j). That is. or a mismatch. and the gap penalty d = 2.2 s <. 0) = −id for the ﬁrst column and F (0. the ﬁnal score F (n. and F (i.nrow=length(y). in case of a match or a mismatch. Dynamic programming of DNA sequences. a match. j) recursively. In particular. It is clarifying to ﬁrst construct the score matrix s(i. 1998. yj ) and in case of a gap we take F (i. For this we use the string-to-character function s2c. m) is the optimal score and the values of the matrix F (i.-1 } rownames(s) <. j) = F (i − 1. we take F (i. j − 1) + s(i. j) − d or F (i. j − 1) + s(xi .184 CHAPTER 9.s2c("GATTA"). j − 1) − d.ncol=length(x)) for (i in 1:(nrow(s))) for (j in 1:(ncol(s))) {if (y[i]==x[j]) s[i. F (i − 1. j) indicates the optimal path. By informaticians this recursive scheme is often called a “dynamic programming algorithm”. we will be able to ﬁnd the best consecutive value for F (i. j) = max F (i − 1. j − 1) − d Note. y <. a for loop. The famous Needleman-Wunsch alignment algorithm consists of taking the maximum out of these possibilities at each step (e. j) = −jd for the ﬁrst row. j) − d  F (i. j − 1). the score +2 for a match. j). and an if else statement. j). al. that this will not yet work because we have not deﬁned any initial values.c(x) > s G A A T T C G 2 -1 -1 -1 -1 -1 A -1 2 2 -1 -1 -1 T -1 -1 -1 2 2 -1 .21). j) F (i.matrix(data=NA. given the values of the previous elements F (i − 1. j) = F (i − 1. Then. Their algorithm can be summarized. Consider again the DNA sequences GAATTC. d <. as follows. -1 for a mismatch.   F (i − 1. Example 2.j]<.g.2 else s[i.s2c("GAATTC"). library(seqinr) x <. j − 1). Durbin et. means that we deﬁne its elements on the basis of the values of its preceding elements. p. GATTA. Building up the matrix F (i. however. In fact we will agree to start with F (0..c(y). ANALYZING SEQUENCES each step we can choose between a gap.j]<. Durbin et. For the two sequences ”PAWHEAE” and ”HEAGAWGHEE” (see. Example 3. Optimal alignment for pairs of amino acid sequences are often considered to be more relevant because these are more closely related to biological functions.d) for (i in 2:(nrow(F))) for (j in 2:(ncol(F))) {F[i.5.j-1]+s[i-1.F[i.j-1]-d))} > F G A A T T C 0 -2 -4 -6 -8 -10 -12 G -2 2 0 -2 -4 -6 -8 A -4 0 4 2 0 -2 -4 T -6 -2 2 3 4 2 0 T -8 -4 0 1 5 6 4 A -10 -6 -2 2 3 4 5 From the lower corner to the right hand side we see that the optimal score is indeed 5.matrix(data=NA.x) F[.names=FALSE)) . using the BLOSUM50 (mis)match score matrix and gap penalty d = 8.matrix(read.] <..nrow=(length(y)+1).21) we seek the Needleman-Wunsch optimal alignment score.j]-d. In particular.1] <. j).F[i-1.y). we shall use the gap penalty d = 8 and for the (mis)match the scores from the so-called BLOSUM50 matrix. Programming Needleman-Wunsch. p.ncol=(length(x)+1)) rownames(F) <.nih.max(c(F[i-1. For this purpose we may modify the previous scheme by changing the gap penalty d and the (mis)match scores s(i. al.c("".ncbi. You can either directly read a BLOSUM matrix from NCBI > > file <. j)."ftp://ftp.as.gov/blast/matrices/BLOSUM50" BLOSUM50 <. F[1. PAIRWISE ALIGNMENTS T -1 -1 -1 2 2 -1 A -1 2 2 -1 -1 -1 185 To initialize the ﬁrst row and column of the matrix F (i. The purpose of the max function seems obvious.j] <.j-1].-seq(0.-seq(0.table(file.length(x)*d. it is convenient to use the function seq.9. check.c("". F <. colnames(F) <.length(y)*d.d). 1998. c("".ncol=(length(x)+1)) F[1.j]-d.-seq(0. library(seqinr).56. y <. For the sake of clarity we shall conveniently construct the matrix s(i.186 CHAPTER 9.x) for (i in 2:(nrow(F))) for (j in 2:(ncol(F))) {F[i.j-1]+s[i-1.max(c(F[i-1.F[i.library(Biostrings).8).1: BLOSUM50 matrix.matrix(data=NA.BLOSUM50[y. ANALYZING SEQUENCES Table 9. A 5 -2 -1 -2 -1 -1 -1 0 -2 -1 -2 -1 -1 -3 -1 1 0 -3 -2 0 R -2 7 -1 -2 -4 1 0 -3 0 -4 -3 3 -2 -3 -3 -1 -1 -3 -1 -3 N -1 -1 7 2 -2 0 0 0 1 -3 -4 0 -2 -4 -2 1 0 -4 -2 -3 D C Q -2 -1 -1 -2 -4 1 2 -2 0 8 -4 0 -4 13 -3 0 -3 7 2 -3 2 -1 -3 -2 -1 -3 1 -4 -2 -3 -4 -2 -2 -1 -3 2 -4 -2 0 -5 -2 -4 -1 -4 -1 0 -1 0 -1 -1 -1 -5 -5 -1 -3 -3 -1 -4 -1 -3 E -1 0 0 2 -3 2 6 -3 0 -4 -3 1 -2 -3 -1 -1 -1 -3 -2 -3 G H I 0 -2 -1 -3 0 -4 0 1 -3 -1 -1 -4 -3 -3 -2 -2 1 -3 -3 0 -4 8 -2 -4 -2 10 -4 -4 -4 5 -4 -3 2 -2 0 -3 -3 -1 2 -4 -1 0 -2 -2 -3 0 -1 -3 -2 -2 -1 -3 -3 -3 -3 2 -1 -4 -4 4 L -2 -3 -4 -4 -2 -2 -3 -4 -3 2 5 -3 3 1 -4 -3 -1 -2 -1 1 K M F P S -1 -1 -3 -1 1 3 -2 -3 -3 -1 0 -2 -4 -2 1 -1 -4 -5 -1 0 -3 -2 -2 -4 -1 2 0 -4 -1 0 1 -2 -3 -1 -1 -2 -3 -4 -2 0 0 -1 -1 -2 -1 -3 2 0 -3 -3 -3 3 1 -4 -3 6 -2 -4 -1 0 -2 7 0 -3 -2 -4 0 8 -4 -3 -1 -3 -4 10 -1 0 -2 -3 -1 5 -1 -1 -2 -1 2 -3 -1 1 -4 -4 -2 0 4 -3 -2 -3 1 -1 -3 -2 T W 0 -3 -1 -3 0 -4 -1 -5 -1 -5 -1 -1 -1 -3 -2 -3 -2 -3 -1 -3 -1 -2 -1 -3 -1 -1 -2 1 -1 -4 2 -4 5 -3 -3 15 -2 2 0 -3 Y -2 -1 -2 -3 -3 -1 -2 -3 2 -1 -1 -2 0 4 -3 -2 -2 2 8 -1 V 0 -3 -3 -4 -1 -3 -3 -4 -4 4 1 -3 1 -1 -3 -2 0 -3 -1 5 A R N D C Q E G H I L K M F P S T W Y V or load a BLOSUM matrix from the Biostrings package. d <.c("".x].8 F <.1] <. s <.nrow=(length(y)+1).j] <.F[i-1.8) rownames(F) <.s2c("PAWHEAE").-seq(0.80.data(BLOSUM50) x <.] <.j-1]. F[. j) without any concern about computer memory.s2c("HEAGAWGHEE"). colnames(F) <.j-1]-d))} .y). data(BLOSUM50) > pairwiseAlignment(AAString("PAWHEAE"). PAIRWISE ALIGNMENTS > F 0 -8 -16 -24 -32 -40 -48 -56 H -8 -2 -10 -18 -14 -22 -30 -38 E -16 -9 -3 -11 -18 -8 -16 -24 A -24 -17 -4 -6 -13 -16 -3 -11 G -32 -25 -12 -7 -8 -16 -11 -6 A -40 -33 -20 -15 -9 -9 -11 -12 W -48 -41 -28 -5 -13 -12 -12 -14 G -56 -49 -36 -13 -7 -15 -12 -15 H -64 -57 -44 -21 -3 -7 -15 -12 E -72 -65 -52 -29 -11 3 -5 -9 E -80 -73 -60 -37 -19 -5 2 1 187 P A W H E A E Hence. p. AAString("HEAGAWGHEE"). Example 5. + scoreOnly = FALSE) Global Pairwise Alignment 1: --P-AW-HEAE 2: HEAGAWGHE-E Score: 1 Hence. seven for y and 10 for x and . from the lower-right corner we observe that the optimal score equals one. library(Biostrings). + substitutionMatrix = "BLOSUM50". gapExtension = -8. we may compute the probability of alignment scores larger than 1. An obvious question is whether in the previous example the obtained score 1 is to be evaluated as being “large” or not. Needleman-Wunsch. To illustrate how the probability of alignment scores larger than 1 can be computed we sample randomly from the names of the amino acids. We may also conveniently use the pairwiseAlignment function from the Biostrings package to ﬁnd the optimal Needleman-Wunsch aligment score for the sequences PAWHEAE” and ”HEAGAWGHEE” (see. Durbin et. 1998.5. A manner to answer this question is by comparing it with the alignment score of random sequences.gapOpening = 0. Example 4.. Comparing with random sequences.9.21). we obtain the optimal score 1 as well as a representation of the optimal alignment. That is. al. replace=TRUE)) randallscore[i] <. scoreOnly = TRUE) > pairwiseAlignment(AAString(y).c2s(ccnd3transl[[1]][50:70]) nwscore <. This is repeated 1000 times and the probability of optimal alignment scores greater than 1 is estimated by the corresponding proportion.double() for (i in 1:1000) { x <. library(seqinr).data(BLOSUM50) randallscore <. The probability of scores larger than 1 equals 0."sp=homo sapiens AND k=ccnd3@") ccnd3 <. choosebank("genbank").gapOpening = 0.003 By the option scoreOnly = TRUE the optimal score is written to the vector randallscore. scoreOnly = TRUE) } > sum(randallscore>1)/1000 [1] 0.substitutionMatrix = "BLOSUM50".7.length(ccnd3transl[[1]]) for (i in 1:(n-21)) nwscore[i] <pairwiseAlignment(AAString(c2s(ccnd3transl[[1]][i:(i+20)])). getTrans) x <. substitutionMatrix = "BLOSUM50". substitutionMatrix = "BLOSUM50".10.library(Biostrings). AAString(y). AAString(y).gapOpening = 0. gapOpening = 0.c2s(sample(rownames(BLOSUM50). n <. We may also program a sliding window such that for each the Needleman-Wunsch alignment score is computed.c2s(sample(rownames(BLOSUM50). library(seqinr) query("ccnd3hs".sapply(ccnd3.003 and is therefore small and the alignment is stronger than expected from randomly constructed sequences. gapExtension = -8. Then the maximum can be found and localized. gapExtension = -8. ANALYZING SEQUENCES compute the maximum alignment score. AAString(y). . gapExtension = -8. replace=TRUE)) y <. Example 6.188 CHAPTER 9. Sliding window on Needleman-Wunsch scores. getSequence) ccnd3transl <.pairwiseAlignment(AAString(x).double() .c2s(ccnd3transl[[1]]) y <.sapply(ccnd3hsreq.

9.6. to translate these and to compute relevant quantities such as the isoelectric point or the hydropathy score.6 Overview and concluding remarks It was illustrated how the query language of the seqinr library can be used to download sequences.9. Furthermore. Dotplot of sequences. the ccnd3 sequences using the query language and write the ﬁrst sequence to a ﬁles in FASTA format. Also try to write them all to FASTA format. The package Biostrings contains the various PAM matrices for optimal alignment. Further applications are given by the exercises below. Writing to a FASTA ﬁle. (b) Construct a plot of the ﬁrst against the ﬁrst and the ﬁrst against the ﬁrst in reverse order. Read. . 2.max(nwscore) [1] 50 189 Note that the maximum occurs when the subsequences are identical. Use the function dotPlot of the seqinr package and par(mfrow=c(1. it was illustated how patterns can be matched and how algorithms for optimal pairwise alignment can be programmed.2)) to produce two adjacent plots. similar to the above. OVERVIEW AND CONCLUDING REMARKS + scoreOnly = TRUE) [1] 152 > max(nwscore) [1] 152 > which. 9. as well as facilities to ﬁnd palindromes. (a) Construct two random sequence of size 100 and plot the ﬁrst against second and the ﬁrst against the ﬁrst.7 Exercises 1. and to read and write data in FASTA format (readFASTA). The value of the maximum is 152 which occurs at position 50.

and MED4 at 5 m..00007%. Construct a dotplot of the most similar and the least similar sequences. and BX548175. The accession numbers of Gen bank are AE017126. optimal alignment is deﬁnes as the maximum over the whole matrix. Sample x and y randomly from the names of the amino acids. It is hypothesized that the G + C content depends on the amount of radiation. respectively. SS120 at 120 m. ANALYZING SEQUENCES (c) Download the sequences related to the species homo sapiens and a gene name like ”CCND3 Cyclin D3”. The latter strain is considered to be high-light-adapted. 3. respectively.190 CHAPTER 9. j) − d  F (i. Each of three strains of P.   F (i − 1. Local alignment. Prochlorococcus marinus. marinus is exposed to diﬀerent intensities of UV radiation because these live in different depths in water.0002% and 70%. Report your observations. The MIT 9313 strain lives at depth 135 m. (b) Compte the GC fraction of each of the sequences. j) = max F (i − 1. 0. . Their algorithm can be summarized (Durbin et al. as follows. 2005.22). The residual intensities of 260-nm UVb irradiation corresponding to the given depths is 0. BX548174. repeat this 1000 times and compute the optimal alignment score and use it to evaluate the signiﬁcance of the previously obtained score. (a) Use the operator OR together with the accession numbers to download the sequences of the bacteria strains. j) F (i. j − 1) + s(i. (c) Is there a relation between UVb radiation and GC fraction? (d) Formulate a relevant hypothesis and test it. 4. j − 1) − d The algorithm allows the score zero if the others have negative values. The Smith-Waterman algorithm seeks maximum local alignment between subsequences of sequences. Probability of more extreme alignment score. seven for y and 10 for x. 5. The idea is that the maximum alignment can occur anywhere in the matrix. p. Program the Smith-Waterman algorithm and ﬁnd the optimal local alignment of the sequences PAWHEAE” and ”HEAGAWGHEE”.

How many do you expect by chance? 9. Use BLOSUM50. Find PR00851A which contains blocks of protein related to a human gene responsible for DNA-repair defect xeroderma pigmentosum (sensitivity to ultraviolet light) Perform a pairwise alignment with these subsequences and report the ones most and least similar. Sequence equality. in what position do they diﬀer? 7. (a) Compute the length of the sequences. (a) Redo the example and brieﬂy describe its usage.fhcrc.uco. if not.7. (b) Translate the sequences into amino acids and compare their frequencies. Plot of CG proportion from Celegans. (c) Are they equal or.ce2) along a window of 100 nucleotides. Download the sequences ”AF517525.CCND3” and ”AL160163. Conserved region. (a) Produce a plot of the CG proportion of the chromosome I of Celegans (Celegans. 8.000 nucleotides. EXERCISES 191 6.CCND3”. At http://blocks.9. (b) A binding sequence of the enzyme EcoRV is the subsequence GATATC. Take the ﬁrst 10. Hint: These are the ﬁrst two from the query ”ccnd3” within homo sapiens. Go to the seqinr help page on dotchart.org there are blocks of highly conserved regions for proteins in PROSITE. How many exact matches has Chromosome I of Celegans. Plot of codon usage.UCSC. (b) Use the query language to ﬁnd .

ANALYZING SEQUENCES .192 CHAPTER 9.

1 193 . Various models for phylogenetic trees are explained in terms of the rate matrix as well as the probability transition matrix. By (Hidden) Markov Models the speciﬁc repetitive order of DNA sequences can be modeled so that predictions of families becomes possible. and models for phylogenetic trees.g. inevitable for the understanding of Markov processes. This chapter is somewhat more technical in its notation with respect to e. The basic ideas of the Hidden Markov Model are brieﬂy explained and illustrated by an example1 . conditional probability. Two basic examples illustrate this point. models for sequence alignment. By the latter it is possible to estimate distances between several sequences and to visualize these in a tree. The latter is the same as a distribution with certain properties. In this chapter you learn what a probability transition matrix is and which role it plays in a Markov process to construct speciﬁc sequences. Recall from Chapter 3 that a discrete distribution is a set of values with certain probabilities that add up to one.Chapter 10 Markov Models The idea of a Markov process forms the basis of many important models in bioinformatics such as (Hidden) Markov Models. This is. 10. Classical matrices for sequence alignment such as BLOSUM and PAM are constructed on the basis of a Markov process. however.1 Random sampling Models to predict and classify DNA type of sequences make it possible to draw a sample from a population.

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Example 1. Throwing a coin. A fair coin X attains Head and Tail with probability 1/2. Thus we may write P (X = H) = 0.5 and P (X = T ) = 0.5. With such a random variable there always correspond a population as well as a sampling scheme which can be simulated on a computer (e.g. Press, et al., 1992). > sample(c("H","T"),30,rep=TRUE,prob=c(0.5,0.5)) [1] "H" "H" "T" "T" "T" "H" "H" "T" "T" "H" "H" "H" "T" "T" "H" "T" [20] "H" "T" "T" "T" "H" "T" "H" "T" "T" "T" "T" Thus the sampled values Head and Tail correspond to the process of actually throwing with a fair coin. The function sample randomly draws thirty times one of the values c("H","T") with replacement (rep=TRUE) and equal probabilities (prob=c(0.5,0.5)). Example 2. Generating a sequence of nucleotides. Another example is that of a random variable X which has the letters of the nucleotides as its values. So the events are X = A, X = C, X = G, and X = T . These events may occur in a certain DNA sequence with probabilities P (X = A) = 0.1, P (X = G) = 0.4, P (X = C) = 0.4, and P (X = T ) = 0.1, respectively. Then the actual placement of the nucleotides along a sequence can be simulated. > sample(c("A","G","C","T"),30,rep=TRUE,prob=c(0.1,0.4,0.4,0.1)) [1] "G" "C" "T" "G" "C" "G" "G" "G" "T" "C" "T" "T" "C" "C" "C" [20] "G" "G" "C" "G" "G" "G" "C" "C" "C" "G" "C" Of course, if you do this again, then the resulting sequence will diﬀer due to the random nature of its generation. For these sampling schemes it holds that the events occur independently from the previous.

10.2

Probability transition matrix

In order to build a model that produces speciﬁc sequences we will consider a certain type of random variable. In particular, we will consider a sequence {X1 , X2 , · · · } with values from a certain state space E. The latter is simply a set containing the possible values or states of a process. If, for instance, Xn = i, then the process is in state i at time n. Similarly, the expression

10.2. PROBABILITY TRANSITION MATRIX

195

P (X1 = i) denotes the probability that the process is in state i at time point 1. The event that the process changes its state from i to j (transition) between time point one and two corresponds to the event (X2 = j|X1 = i), where the bar means ”given that”. The probability for this event to happen is denoted by P (X2 = j|X1 = i). In general, the probability of the transition from i to j between time point n and n + 1 is given by P (Xn+1 = j|Xn = i). These probabilities can be collected in a probability transition matrix P with elements pij = P (Xn+1 = j|Xn = i). We will assume that the transition probabilities are the same for all time points so that there is no time index needed on the left hand side. Given that the process Xn is in a certain state, the corresponding row of the transition matrix contains the distribution of Xn+1 , implying that the sum of the probabilities over all possible states equals one. The probability transition matrix contains a (conditional) discrete probability distribution on each of its rows. For a Markov process it holds that the state at time point n + 1 depends upon the state at time point n, but not on states at earlier time points. Example 1. Using the probability transition matrix to generate a Markov sequence. Suppose Xn has two states: 1 for a pyrimidine and 2 for a purine. A sequence can now be generated, as follows. If Xn = 1, then we throw with a fair die: If the outcome is lower than or equal to 5, then Xn+1 = 1 and, otherwise, (outcome equals 6) Xn+1 = 2. If Xn = 2, then we throw with a fair coin: If the outcome equals Tail, then Xn+1 = 1, and otherwise Xn+1 = 2. For this process the two by two probability transition matrix equals to 1 2 from , 1 p11 p12 2 p21 p22 where p21 is the probability that the process changes from 2 to 1. This transition matrix can also be written as P = p11 p12 p21 p22 = P (X1 = 1|X0 = 1) P (X1 = 2|X0 = 1) P (X1 = 1|X0 = 2) P (X1 = 2|X0 = 2) =
5 6 1 2 1 6 1 2

.

Any matrix probability transition matrix P can be visualized by a transition graph, where the transition probabilities are visualized by an arrow from

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state i to state j and the value of pij . For the current example the transition graph is given by Figure 10.12 . The values 1 and 2 of the process are written within the circles and the transition probabilities are written near the arrows. To actually generate a sequences with values equal to 1 and 2 according the
1/6

1/2 0 5/6 1

1/2

Figure 10.1: Graph of probability transition matrix transition matrix we may use the following. markov1 <- function(x,P,n){ seq <- x for(k in 1:(n-1)){ seq[k+1] <- sample(x, 1, replace=TRUE, P[seq[k],])} return(seq) } P <- matrix(c(1/6,5/6,0.5,0.5), 2, 2, byrow=TRUE) rownames(P) <- colnames(P) <- StateSpace <- x <- c(1,2) > markov1(x,P,30) [1] 1 2 1 2 1 2 2 1 2 1 2 2 1 2 1 2 2 1 2 1 2 1 2 1 2 2 2 2 2 2 In the function markov1 the actual sampling is conducted by sample. We sample one element from the set containing 1 and 2 according to the probabilities in row seq[k] of the matrix P. This makes the probabilities of the states dependent on the corresponding row of the transition matrix. We conveniently use the fact that R adds an element to the sequence; we do not have to declare its length on beforehand (although we could!). The sequence has a ﬁxed start at State 1 and thereafter the ﬁrst row in the probability transition matrix. Note that without the return command the function does
2

The values 1 and 2 are erroneously depicted as 0 and 1, respectively

10.2. PROBABILITY TRANSITION MATRIX not give any output.

197

Example 2. A sequence with a large frequency of C and G. To illustrate that certain probability transition matrices imply a large frequency of C and G residues, we use the following. markov2 <- function(StateSpace,P,pi0,n){ seq <- character(n) seq[1] <- sample(StateSpace, 1, replace=TRUE, pi0) for(k in 1:(n-1)){ seq[k+1] <- sample(StateSpace, 1, replace=TRUE, P[seq[k],])} return(seq) } P <- matrix(c( 1/6,5/6,0,0, 1/8,2/4,1/4,1/8, 0,2/6,3/6,1/6, 0,1/6,3/6,2/6),4,4,byrow=TRUE) rownames(P) <- colnames(P) <- StateSpace <- c("a","c","g","t") pi0 <- c(1/4,1/4,1/4,1/4) x <- markov2(StateSpace,P,pi0,1000) > table(x) x a c g t 72 409 378 141 The function starts with sampling just once from the distribution with equal probabilities pi0. It conveniently uses the the column and row names of the probability transition matrix for the sampling. The probabilities to go from ”a” or ”t” to ”c” or ”g” are large and as well as that to stay within ”c” or ”g”. From the frequency table it can be observed that the majority of residues are ”c” or ”g”. Example 3. A sequence with high phenylalanine frequency. Now it is possible to construct a sequence which produces the amino acid phenylalanine (F) with high probability. Recall that it is coded by the triple TTT or TTC. We use the function getTrans of the seqinr package to translate nucleotide triplets into amino acids.

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CHAPTER 10. MARKOV MODELS

pi0 <- c(1/4,1/4,1/4,1/4) P <- matrix(c(.01,.01,.01,.97, .01,.01,.01,.97, .01,.01,.01,.97, .01,.28,.01,0.70),4,4,byrow=T) rownames(P) <- colnames(P) <- StateSpace <- c("a","c","g","t") x <- markov2(StateSpace,P,pi0,30000) > table(getTrans(x)) * 2 Y 60 A 1 C 75 D F 2 5205 H 24 I L 76 2260 M 1 N 2 P 19 R S 26 2154 T 1 V 91

From the table it is clear that the F frequency is the largest among the generated amino acids. Example 4. To illustrate estimation of the probability transition matrix we proceed with the sequence produced by the previous example. nr <- count(x,2) names(nr) A <- matrix(NA,4,4) A[1,1]<-nr["aa"]; A[1,2]<-nr["ag"]; A[1,3]<-nr["ac"]; A[1,4]<-nr["at"] A[2,1]<-nr["ga"]; A[2,2]<-nr["gg"]; A[2,3]<-nr["gc"]; A[2,4]<-nr["gt"] A[3,1]<-nr["ca"]; A[3,2]<-nr["cg"]; A[3,3]<-nr["cc"]; A[3,4]<-nr["ct"] A[4,1]<-nr["ta"]; A[4,2]<-nr["tg"]; A[4,3]<-nr["tc"]; A[4,4]<-nr["tt"] rowsumA <- apply(A, 1, sum) Phat <- sweep(A, 1, rowsumA, FUN="/") rownames(Phat) <- colnames(Phat) <- c("a","g","c","t") > round(Phat,3) a g c t a 0.011 0.000 0.007 0.982 g 0.017 0.003 0.010 0.969 c 0.010 0.011 0.012 0.967 t 0.009 0.009 0.279 0.703 The number of transitions are counted and divided by the row totals. The estimated transition probabilities are quite close to the true transition probabilities. The zero transition probabilities are exactly equal to the true because

3 Properties of the transition matrix In the above.2) n n+1 Thus to obtain the probabilities of the states at time point n + 1. Note that the last equality holds by deﬁnition of matrix multiplication.P = 5 6 1 2 1 6 1 2 . 10. where 0 1 π T = (P (X1 = 1).10. where p2 is column 2 of the 0 transition matrix P = (p1 . it can be shown that P (X1 = 2) = π T p2 . It can be concluded that π T P = π T .7. that is πT P = πT . Often. (10. Example 1. . see Section 10. the probability at time point 1 that the 1 process is in State 1. (10. This estimation procedure can easily be applied to DNA sequences. Then P (X1 = 1) and P (X1 = 2) collected in π T = (P (X1 = 1). the sequence was started at a certain state. we have a vector π 0 with initial probabilities π10 = P (X0 = 1) and π20 = P (X0 = 2). 1 πT = πT P = 1 0 3 2 3 1 3 T 5 6 1 2 1 6 1 2 = 2 3 ·5+1· 6 3 1 2 2 3 ·1+1· 6 3 1 2 = 13 18 5 18 The transposition sign simply transforms a column into a row. PROPERTIES OF THE TRANSITION MATRIX 199 these do not occur. In a similar manner. Suppose the following initial distribution and probability matrix π0 = 2 3 1 3 .3. for State 1 and 2. respectively. p2 ). however. Matrix multiplication to compute probabilities. State 2. then the probability that the process is in State 1 at time point 1 can be written as P (X1 = 1) = π10 p11 + π20 p21 = π T p1 . Furthermore. That is. the probabilities of the initial states are available.1) 0 where p1 is the ﬁrst column of P . P (X1 = 2)) can be computed as follows. respectively. P (X1 = 2)). if the transition matrix P = p11 p12 p21 p22 = P (X1 = 1|X0 = 1) P (X1 = 2|X0 = 1) P (X1 = 1|X0 = 2) P (X1 = 2|X0 = 2) . This holds in general for all time points n. we can simply use matrix multiplication 3 .

c(2/3.1/6. j can be computed by matrix multiplication. we have that P (Xn = j|X0 = i) = pn .2] [1. p. j of P n .0. it holds (see Section 10. Example 3.7222222 0.3333333 Larger powers of P can be computed more eﬃciently by methods given below. the values P (X2 = j|X0 = i) for all of i. 4 .matrix(c(5/6. another important property of the probability transition matrix deals with the probability of being in state 1 given that the process is in state 1 two time points before.2. such matrix multiplications can be accomplished much more convenient on a personal computer.byrow=T) > pi0 <. 11 (10.5).2] [1.7) that P (X2 = 1|X0 = 1) = p2 . see Pevsner (2003.1/3) > pi0 %*% P [. In particular. MARKOV MODELS Using R its operator %*% for matrix multiplication.200 CHAPTER 10. Obviously.5.1] [.2777778 Yet. > P <. > P %*% P [. the product π T P can be 0 computed as follows.2222222 [2. In general. Given the probability matrix of the previous example.] 0.6666667 0. 1) of the matrix4 P 2 .2.0.56) or wikipedia using the search string ”wiki matrix multiplication”.] 0.7777778 0. For a brief deﬁnition of matrix multiplication.1] [.3) where the latter is element (1.] 0. ij which is element i. P2 = 5 6 1 2 1 6 1 2 · 5 6 1 2 1 6 1 2 = ( 5 )2 + 1 1 6 62 15 + ( 1 )2 26 2 51 + 11 66 62 11 + ( 1 )2 26 2 = 28 36 24 36 8 36 12 36 .

In other words. For any initial distribution π 0 .197) because a probability transition matrix has a unique largest eigenvalue equal to 1 with corresponding eigenvectors 1 and π (or rather normalized versions of these). as follows P 3 = V ΛV −1 V ΛV −1 V ΛV −1 = V ΛΛΛV −1 = V Λ3 V −1 . which is equal to element j of π. given that it started in State 1. It follows that. So that. it follows that π 0 P n tends to π T . In the long term the Markov process tends to a certain value (Br´maud. then P n tends to 1π T .4 Stationary distribution πT = πT P A probability distribution π satisfying is stationary because the transition matrix does not change the probabilities of the states of the process. Stationary distribution. is unique.4. p. j) of 1π T .10. The latter is a computationally convenient expression because we only have to take the power of the eigenvalues in Λ and to multiply by the left and right eigenvector matrices. To compute these we need the eigen-decomposition of the probability transition matrix P = V ΛV −1 . P (Xn = j|X0 = i) = pn tends to ij element (i. It sheds light on the question: What is the probability P (Xn = 1|X0 = 1) = pn . This will be illustrated below. STATIONARY DISTRIBUTION 201 10. e 1999. Such a distribution usually exists. as n 11 increases without bound. To compute the eigen-decomposition of the probability transition matrix P as well as powers of it. where V is the eigenvector matrix and Λ the diagonal matrix with eigenvalues. as time increases without bound? To answer such a question we need large powers of the probability transition matrix. and plays an essential role in the long term behavior of the process. Example 1. indeed. . That is: What is the probability that the process is in State 1. as n increases without bound. in general P n = V Λn V −1 . Now the third power of the probability transition matrix can be computed. we may use the function eigen. The latter are usually sorted in decreasing order so that the ﬁrst (left upper) is the largest.

3] [1. > V$vec %*% diag(V$va)^(16) %*% solve(V$vec) [.3333333 > V$vectors [.625 So that the stationary distribution π T equals (0.matrix(c(1/6.1/2. When we consider pure self-fertilization. 0) for the events (AA. that of aa is aa with probability (0. 1). Suppose A is a dominant gene.0.2.202 CHAPTER 10.3.0.] -0. 0.byrow=T) V <. aA.1] [.2] [1.625).1).byrow=T) > V <.5.1/4. P <. then the oﬀspring from AA is AA with probability (1.000000 0. 0. the probability transition matrix raised to the power sixteen. aA.5).] 1. 1/2. 1.2] [1.375 0. and that of aA is (AA.0. 0.00000 0. 1/4.0.8574929 [2.625 [2.375 0.5144958 The output of the function eigen is assigned to the list V from which the eigenvalues and eigenvectors can be extracted and printed to the screen.matrix(c(1.] 0.0. 1/4. 0). the probability transition matrix becomes   1 0 0 P =  1/4 1/2 1/4  0 0 1 We can now compute the transition probability matrix after ﬁve generations.] 0.symmetric = FALSE) > V$vec %*% diag(V$va)^(5) %*% solve(V$vec) [.eigen(P.7071068 0. Example 2.375. a a recessive and that we start with a heterozygote aA.eigen(P. From the latter we obtain the initial state probability π T = (0.000000 . respectively.2.2] [.] -0.3.0. Now we can compute P 16 . aa) with probability 1/4.1] [. Diploid.5/6.7071068 -0.symmetric = FALSE) > V$values [1] 1. aa).1] [.0000000 -0. MARKOV MODELS > P <. Hence.

20 A into C. From a rate matrix to a probability transition matrix. Deonier.000000 203 Hence.20  0. Note that this method of raising the transition probability matrix to a large power can easily be applied to determine the stationary distribution.484375 [3. PHYLOGENETIC DISTANCE [2. 1/2). using Equation 10.20 0.484375 0.20 −0. and 0. 0. 2005. How to do this in practice will be illustrated by an example. Example 1.20 0. p. a proportion of 0.20 0.03125 0.20 −0.60 of the .20 −0. The idea of taking a transition matrix to a certain power is also used to construct the PAM250 matrix given the PAM1 matrix (Pevsner.20 0.53) and for the construction of various BLOSUM matrices (Pevsner. Consequently. .20 A changes into G. et al.10. − . These distances are computed from substitution models which are deﬁned by a matrix representing the rate of substitutions of one state to the other.20 T 0. The probability transition matrix P can be computed by matrix exponentiation P = exp(Q). it can be shown that n n n 1 1 1 1 1 πT = .] 0.5059.00000 1.20 A into T . The rates of staying in a state are given by a negative number on the diagonal of the substitution matrix.60 0.000000 0. The latter is usually expressed as a matrix Q.20 0. p.20 Q= G  C  0.   Thus within a certain time period a proportion of 0.20 0. A little more precise. 187-190). 2003.2. Suppose the rate matrix A G C T  A −0. 2003. the distribution we obtain can be read from the second row which is highly homozygotic.60   .] 0. 10.5 Phylogenetic distance Phylogenetic trees are constructed on the basis of distances between DNA sequences.5.60 0.60 0. 0. − n+1 2 2 2 2 2 so that the distribution converges to (1/2.

Because all phylogenetic models are deﬁned in terms of rate matrices.1.-3.1.14 T 0.1. we shall concentrate on these. G ↔ T . This assumption.14.A ↔ C.14 0. etc.59 0.14 0. Furthermore. the change from i to j equals that from j to i. so that the rate matrix is symmetric.14 0. is unrealistic is many cases.0.   QJC69 The sum of each row of a rate matrix equals zero. from A to G is 0.1.2) A G C T A 0.1.59 0.colnames(Q) <. That is.matrix(expm(Q)) > round(P.14 0.59 0. however.59 Thus the probability that the state changes from A to A is 0.2 * Matrix(c(-3. library(Matrix) Q <.14 0. and C ↔ G).1. Transitions are substitutions of nucleotides within types of nucleotides.14 0."T") P <.4) rownames(Q) <. Also the probability that the sequence equals one of the nucleotides is 1/4. we can ﬁnd the probability transition matrix P = exp(Q) by using the function expm(Q) from the package Matrix.59.as.1.204 CHAPTER 10. so that from this requirement the diagonal elements of the JC69 model are equal to −3α.1. MARKOV MODELS residues goes back to A. thus purine to purine or pyrmidine to pyrmidine (A ↔ G or C ↔ T ).c("A".14 0.14 0. the non-diagonal substitution rates of the JC69 model all have the same value α. Given this rate matrix.-3)."C".14 G 0. Transversions are substitutions between nucleotide type (A ↔ T . the rate matrix for the Jukes and Cantor (1969) (JC69) model can be written as A A · = G  α  C  α T α G α · α α C α α · α T α α α ·   .1.1.14 0.-3.1."G".14 C 0. In the JC69 model a transition is assumed to . For instance.1.

3] <. then the number of transversions is small. which are commonly abbreviated by K80 and K81.  β γ · α . γ = 1/6 we may use the following. For instance.4) Q[1. the rate of change A → G is α and equals that of A ← G.2] <. Some examples of models with even more parameters are the Hasegawa. Example 2. gamma<. alpha <. In the K81 model all changes occur at a diﬀerent though symmetric rate. The K81 model. if both β and γ are very small. it does not account for the fact that transitions are more common that transversions. 1981). From these distances the phylogenetic tree is computed by a neighbor-joining algorithm such that it has the smallest total branch length.1/6.Q[4.1] <. then the amount of transitions is large.matrix(data=NA. Similarly.alpha   . then we obtain the JC69 model. beta <. If α is large. β = 2/6.3/6.10. PHYLOGENETIC DISTANCE 205 happen with equal probability as a transversion. To cover this for more general type of models are proposed by Kimura (1980. Kishino.5.  .4.Q[2. Q <. In terms of the rate matrix these models can we written as     · α β β · α β γ  α · β β   α · γ β    QK80 =  QK81 =   β β · α . the JC69 model is nested in the K80 model because if we take β = α. then we obtain the K80 model. To compute the rate matrix of the K81 model with α = 3/6. A model is called “nested” if it is a special case of a more general model. and Yano (1985) (HKY85) model and the General Time-Reversable Model (GTR) model    · απG βπC βπT · απG βπC γπT  απA   απA · βπC βπT  · δπC πT QHKY 85 =  QGT R =   βπA βπG .2/6. β β α · γ β α · In the K80 model a change within type (transition) occurs at rate α and between type (transversion) at rate β. That is.4] <.  βπA δπG · απT · ζπT βπA βπG απC · γπA πG ζπC · The distance between DNA sequences is deﬁned on the basis of these models. the K80 model is nested in the K81 model because when we take γ = β.Q[3.

2288517 0.16).2] [.25.5000000 -1.1393498 0.0000000 0.1666667 -1.25 0.Matrix(Q) P <.Q[3.3333333 0.3333333 0.0000000 > Q <.25 0. library(Matrix) alpha <.1] [.5000000 -1.0000000 0.] 0. Stationarity for the JC69 model.5000000 [4.] 0.4] [1.1767105 0.-3 * alpha Q <.1767105 [3.1393498 0. Q <. it can be observed that the stationary distribution π T = (0.1393498 [2.25 0.eigen(P.2288517 0.25 0.206 CHAPTER 10.] -1.2288517 0. and raise it to the power 50.] 0.25 [2.Q[4. Example 3.1666667 0.25 0.4550880 0.25 0.1393498 0.Q[3.4.Q[2.3] [.as.2] [.4550880 0.4] [1.2288517 [4. the corresponding probability transitionmatrix P .4] <.matrix(expm(Q)) V <.25 0.matrix(rep(alpha.1666667 0.25 0.Q[2.1] [.25).] 0.] 0.symmetric = FALSE) > V$vec %*% diag(V$va)^(50) %*% solve(V$vec) [.] 0.5000000 0.as.2] [.-(alpha + beta + gamma) > Q [.25.1767105 0.1] <.1] [.1/5.] 0.] 0.gamma > diag(Q) <.3333333 0.Q[4. 0.matrix(expm(Q)) > P [.] 0.] 0.25 [3.1] <.3] <.3] [.3] [. Let’s take α = 1/5 as in Example 1 and compute the rate matrix Q of the JC69 model.1767105 0.1666667 [2.3333333 [3.25 .2] <.25.4550880 By raising the power of the probability transition matrix to a suﬃciently large number.25 0.25 [4. 0.25 0.25 0.4) diag(Q) <.4] [1.] 0.3] <.4] <. MARKOV MODELS Q[1.Matrix(Q) > P <.beta Q[1.2] <. 0.0000000 0.25 0.4550880 0. species. In case of the JC69 model. This can be veriﬁed as follows. 0. Example 4.1326839 Hence. PHYLOGENETIC DISTANCE 207 Hence. 0. species.read.read. The pairwise distances between DNA sequences can be computed by the function dist. the distance is 0. the distance between sequences is a function of the proportion of diﬀerent nucleotides.paste("AJ5345". The function paste is used to quickly deﬁne the accession numbers and read. Inserting this into the previous distance formula gives the distance.133. 0.26:27. model = "JC69") AJ534526 AJ534527 0.GenBank(accnr.25.-log(1-4*p/3)*3/4 > d [1] 0.sum(seq$AJ534526==seqAJ534527)/1143 > d <.10. 2006). We shall use the dist. the stationary distribution is π T = (0.GenBank to actually download the sequences.sep="") seqbin <.dna from the ape package.1326839 Example 5.names = TRUE. Ewens & Grant.character = FALSE) dist. p.character = TRUE) > p <. som that the proportion of diﬀerent nucleotides 139/1143 = p. To further illustrate distances between DNA sequences we shall download the Chamaea fasciata mitochondrial cytb gene for cytochrome b for 10 species of warblers of the genus sylvia (Paradis. 4 where p is the proportion of diﬀerent nucleotides of the two sequences.library(seqinr) accnr <. as. 3 d = − log(1 − 4p/3).25.dna function with the K80 model.25) (cf.25. .5. as.GenBank(accnr. > seq <. Phylogenetic tree of a series of downloaded sequences. 2005. > > > > library(ape).dna(seqbin. 477).names = TRUE. Distance between two sequences according to the JC69 model. Namely. The species names are extracted and attached to the sequences. Over a total of 1143 nucleotides there are 139 diﬀerences. 966 9213.931 JC69+I 2 -4425.attr(seq.608 JC69+I+G 3 -4421.079 K80+G 3 -4224.dist.915 K80+I+G 4 -4223. Example 6.para loglik AIC JC69 1 -4605.txt".457 8454.662 .455 K80+I 3 -4230. then the Akaike information criterion (AIC = -2 · loglik + 2 · number of free parameters) can be used to select models. When the models are estimated by maximum likelihood. The best model is the one with the smallest AIC value. "species") dist <.txt".lirmm. execname ="phyml_linux") print(out) nb. The output from the program is written to the object called out for which the functions plot(out) and summary(out) can be used to extract more detailed information.sep="") seq <.paste("AJ5345".208 CHAPTER 10. > > > > setwd("/share/home/wim/bin") write. in this manner various trees can be computed and their plots compared. model = "K80") plot(nj(dist)) Obviously.331 9036.read.000 8848. When various diﬀerent models are deﬁned the question becomes apparent which of these ﬁts best to the data relative to the number of parameters (symbols) of the model.272 F81 4 -4514.dna(seq.727 8851.library(seqinr) accnr <. 2003) can be downloaded from http://atgc.203 JC69+G 2 -4421.602 8855. if the executable is available at the same directory.001 K80 2 -4423."seq.free.format = "interleaved". MARKOV MODELS library(ape). format ="interleaved") out <-phymltest("seq. A program called PHYML (Guindon & Gascuel.304 8846.dna(seq. We ﬁrst write the sequences to the appropriate directory.136 8454.26:35.fr/phyml/ and run by the R function phymltest.539 8467.GenBank(accnr) names(seq) <. 086 -4102. and.01) In case similar sequences have slightly diﬀerent lengths.600 -4304.581 8208.580 8604.txt") plot(tr[[27]]) add.164 -4112.171 8216.020 8178. HIDDEN MARKOV MODELS F81+I F81+G F81+I+G F84 F84+I F84+G F84+I+G HKY85 HKY85+I HKY85+G HKY85+I+G TN93 TN93+I TN93+G TN93+I+G GTR GTR+I GTR+G GTR+I+G 5 5 6 5 6 6 7 5 6 6 7 6 7 7 8 9 10 10 11 -4309.500 -4333. next.624 -4323.012 8225.760 -4351.530 -4303.802 8207.006 -4106.043 8178.524 8206.6 Hidden Markov Models In a Hidden Markov Model (HMM) there are two probability transition matrices.scale. To plot it. these have to be aligned by programs such as clustalx or clustalw before these can be used.3.199 8619. It can be seen that the smallest AIC corresponds to model 27 called GTR+G.bar(length=0. 10.060 8619.291 -4097.299 209 The notation ”+I” and ”+G” indicates the presence of invariant sites and/or a gamma distribution of substitution rates.135 8225.149 8629. The . to extract the 27th.922 8197. see Figure 10.10.461 -4090.6.519 8712.read.398 -4084.010 -4078.txt_phyml_tree.795 8189.328 8236.568 -4105. There is an emission matrix E and a transition matrix A.522 -4079.401 -4096.248 8658. tr <.262 -4097.198 8196.001 8676.tree("seq. we have to read the trees.099 -4091.790 -4293. 10 and that it stays loaded is 0.n){ observationset <. It is convenient to denote fair by 1 and unfair by 2. the probability of outcome 6 equals 1/2 and that of any other outcome equals 1/10. A = = P (Oi = 1|Di = 1) P (Oi = 2|Di = 1) P (Oi = 3|Di = 1) · · · P (Oi = 1|Di = 2) P (Oi = 2|Di = 2) P (Oi = 3|Di = 2) · · · 1/6 1/6 1/6 1/6 1/6 1/6 1/10 1/10 1/10 1/10 1/10 1/2 .90 .10 0. The state with respect to fairness is hidden for the observer. After sampling the hidden states from a Markov chain and the outcomes of the die are sampled according to the value of the hidden state (die type).2) x <. hmmdat <.E. Example 1. MARKOV MODELS generation of an observable sequence goes in two steps. 1998. First.matrix(NA.nr=n. al.nc=1) . given that it is fair at time point i − 1. We shall illustrate this by the classical example of the occasionally dishonest casino (Durbin et.90. The HMM with this transition and emission matrix can be programmed. however.c(1:6) hiddenset <. A casino uses a fair die most of the time. (10. Occasionally dishonest casino. there is a transition from a Markov process of a hidden state and given this value there is an emission of an observable value. The observer can only observe the values of the die and not its hidden state with respect to its fairness. With this emission matrix we can generate a sequence of hidden states. However. given that the die is unfair (loaded).05.c(1. Given the fairness of the die we deﬁne the probability transition matrix. The probability that it will switch from fair to unfair is 0.05 0.18). where the values 1 and 2 indicate whether the die is fair (1) or loaded (2). occasionally it switches to an unfair die.95 0.210 CHAPTER 10.function(A.95 that the die is fair at time point i. the probability of any outcome equals 1/6.4) Thus given that the die is fair. The transition probabilities of the hidden states are by the emission matrix E= P (Di = 1|Di−1 = 1) P (Di = 2|Di−1 = 1) P (Di = 1|Di−1 = 2) P (Di = 2|Di−1 = 2) = 0. p.h <.. The probability that it will switch from loaded to fair is 0. Thus the probability is 0. 2.0. and v(1.10.E.E[h[1]. l) = e(l.1. l). l) = 0 for all l. x[1]<-sample(observationset.h).replace=T. l)} .6.9).replace=T.95.nrow=n.matrix(c(x.n) for(k in 1:(n-1)){x[k+1] <.byrow=T) #emission matrix A <.ncol=2.6.hmmdat(A.markov(hiddenset. where i runs from one to the number of observations and l from one to the number of states.1:100 > t(dat) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 observations 5 2 3 1 6 1 3 1 1 5 6 6 2 2 3 5 4 6 1 2 4 4 3 hidden_states 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 observations 4 3 2 4 1 6 6 6 6 6 5 5 3 6 1 6 5 2 4 1 hidden_states 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 observations 5 6 5 2 3 3 1 3 3 5 6 6 2 4 5 4 6 1 6 5 hidden_states 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 observations 1 1 4 4 1 5 6 4 3 5 4 2 6 1 3 6 5 2 2 6 hidden_states 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 92 93 94 95 96 97 98 99 100 observations 4 1 6 5 5 6 5 3 4 hidden_states 1 1 1 1 1 1 1 1 1 24 2 1 46 4 1 68 2 1 90 6 1 25 3 1 47 2 1 69 6 1 91 1 1 In certain applications to bioinformatics.05.sample(observationset.100) colnames(dat) <.])} out <. k .2.6).byrow=FALSE) return(out) } E <. The algorithm builds up a matrix v(i.A.matrix(c(0.]) h <. The initial values are v(1. x(i)) · max {v(i − 1. l) are recursively deﬁned by v(i. it is of most importance to estimate the value of the hidden state given the data.0.1.c("observation".rep(1/10. Then the values for v(i.1/2).byrow=TRUE) #transition matrix dat <.E[h[k]. k)a(k.2.1.5). HIDDEN MARKOV MODELS 211 h[1]<-1."hidden_state") rownames(dat) <. 1) = 1.0. The Viterbi algorithm is developed to predict the hidden state given the data and the (estimated) transition and emission matrix.matrix(c(rep(1/6. dat[. function(x) which. vitrowmax) vitrowmax hiddenstate 1 2 1 72 11 2 15 2 datt <.2] > table(hiddenstate.matrix(NA.viterbi(A. nr=length(x).cbind(dat.0.x) { v <.1] <.function(A.max(x)) hiddenstate <.1 for(i in 2:length(x)) { for (l in 1:dim(A)[1]) {v[i.E. v[1.] * A[l.] <.x[i]] * max(v[(i-1).E.apply(vit.l] <.c("observation".212 CHAPTER 10. Example 2. nc=dim(A)[1]) v[1.dat[. 1.])} } return(v) } vit <."hidden_state".vitrowmax) colnames(datt) <."predicted state") > t(datt) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 observation 5 2 3 1 6 1 3 1 1 5 6 6 2 2 3 5 4 6 1 2 4 4 3 2 hidden_state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 predicted state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 observation 3 4 3 2 4 1 6 6 6 6 6 5 5 3 6 1 6 5 2 4 1 hidden_state 1 1 1 1 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 predicted state 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 observation 4 2 5 6 5 2 3 3 1 3 3 5 6 6 2 4 5 4 6 1 6 hidden_state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 .E[l.1]) vitrowmax <. viterbi <. MARKOV MODELS For each row of the matrix the maximum is taken as the best predictor of the hidden state. The viterbi algorithm can be programmed and applied to the hidden states of the data generated with respect to the occasionally dishonest casino. X0 = 1) + P (X1 = 1. APPENDIX predicted state 213 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 observation 5 2 6 1 1 4 4 1 5 6 4 3 5 4 2 6 1 3 6 5 2 hidden_state 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 predicted state 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 88 89 90 91 92 93 94 95 96 97 98 99 100 observation 2 6 6 1 4 1 6 5 5 6 5 3 4 hidden_state 2 2 1 1 1 1 1 1 1 1 1 1 1 predicted state 1 1 1 1 1 1 1 1 1 1 1 1 1 The misclassiﬁcation rate is 0.27 which is quite large given the fact that we used the true transition and emission matrix. 10.7. . This is caused by the recursive nature of the algorithm. X0 = 2) P (X1 = 1|X0 = 1) · P (X0 = 1) + P (X1 = 1|X0 = 2) · P (X0 = 2) π10 p11 + π20 p21 π T p1 .10. it takes a few values for the prediction to change. 0 where p1 is the ﬁrst column of P .7 Appendix The probability that the process is in State 1 at time point 1 can be computed as follows. An important observation is that after a transition of a hidden state. P (X1 = 1) = = = = P (X1 = 1. 11 where the latter is element (1.8 Overview and concluding remarks The probability transition matrix is extensively explained and illustrated because it is a cornerstone to many ideas in bioinformatics. and C. T. X1 = 1|X0 = 1) + P (X2 = 1. Computing probabilities. X0 = 1) · P (X1 = k|X0 = 1) k=1 2 = = k=1 P (X2 = 1|X1 = k) · P (X1 = k|X0 = 1) = p11 p11 + p21 p12 = row 1 of P times column 1 of P = P 2 . X1 = 2|X0 = 1) 2 = k=1 2 P (X2 = 1. al (2005). 1) of the matrix P 2 = P · P . 6 6 5 2 3 1  0 2 0 1 1 0 0   0 0 7 7 4 4 7 7 1 1 2 4 5 0 0 3 8 8 8 8 8 8 2. . . MARKOV MODELS P (X2 = 1|X0 = 1) = P (X2 = 1.214 In particular. Given the states 0 and 1 and the following initial distribution and probability matrix π0 = 1 2 1 2 . it holds that CHAPTER 10.9 Exercises For the   . A thorough treatment of phylogenetics is given by Paradis (2006) and of Hidden Markov Models by Durbin et. 10. Visualize by a transition graph the following transition matrices. 10. 6 6 5 6 . . G.  1 2   1 3 0 1 0 0 4 4 4 4 4 1 2  1 2 2 1   1 5 0 0 1 0 0 1 3 3 6 .P = 3 4 1 2 1 4 1 2 .  1. X1 = k|X0 = 1) P (X2 = 1|X1 = k. the process with four states take the names of the nucleotides in order A. 35.10.4.character = TRUE in the read. (c) Compute P (X2 = 0|X0 = 0).9. (b) Is the transversion rate larger or smaller then the transition rate? (c) Compute the corresponding the probability transition matrix. = 0. πT = 0. EXERCISES (a) Compute P (X1 = 0).2.5. (d) Try to argue whether you expect a large frequency of transversions or translations. Hint: Use as. (b) Compute P (X1 = 1). (e) Generate a sequence of 99 nucleotide residues according to the markov model. δ = 0. (d) Compute P (X2 = 1|X0 = 0). Distance according to JC69. (c) Use this proportion to verify the distances between these sequences according to the JC69 model.15. . 215 3. γ = 0. β = 0. (a) Down load the sequences AJ534526 and AJ534527. Programming GTR. (b) Compute the proportion of diﬀerent nucleotides. Use πA = 0. (a) Program the rate matrix in such a manner that it is simple to adapt for other values of the parameters.15. and ζ = 4. πG = 0. α = 4. πC = 0.3. 4.35.GenBank function. 216 CHAPTER 10. .2: Evaluation of models by AIC . MARKOV MODELS Akaike information criterion for phymlout JC69 F81 JC69 + I K80 JC69 + I + Γ JC69 + Γ F84 HKY85 TN93 F81 + I F81 + I + Γ F81 + Γ GTR K80 + I K80 + Γ K80 + I + Γ F84 + I F84 + Γ F84 + I + Γ HKY85 + I TN93 + I HKY85 + I + Γ HKY85 + Γ TN93 + I + Γ TN93 + Γ GTR + I GTR + I + Γ GTR + Γ 9200 9000 8800 8600 8400 8200 Figure 10. 01 Chamaea fasciata Figure 10. .3: Tree according to GTR model.9. EXERCISES 217 Sylvia crassirostris Sylvia hortensis Sylvia leucomelaena Sylvia lugens Sylvia buryi Sylvia boehmi Sylvia subcaeruleum Sylvia layardi Sylvia nisoria 0.10. MARKOV MODELS .218 CHAPTER 10. (a) matrix. number of rows. numeric. set the working directory to a certain map. function.1. function. (c) Use R its help or use the internet search key ”r wiki grep” to ﬁnd the following answers: searching regular expressions. Some questions to orientate yourself. generate a factor by specifying the pattern of levels. matrix. sequence. make R reading input from a ﬁle or URL. numeric. (b) remove. ExpressionSet. standard deviation. sdexprsval <. give the structure of an object.order(sdexprsval.sd).2. 2. (c) To order the data frame according to the gene standard deviations. gendat (a) apply(gendat.decreasing=TRUE) gendat[o.Appendix A Answers to exercises Answers to exercises of Chapter 1: Brief Introduction to R 1. standardGeneric.1.apply(gendat. print the last · commands given from the command line.sd) o <.sd). (b) apply(gendat. product. return a vector from a function on the rows or columns of a matrix. factor.] 219 . summation. load addon packages. 5.golub.gnames[.sd).1. Computations on gene means of the Golub data.220 (d) gene1 APPENDIX A. "X01677_f_at" > golub.5) gives that the number of genes having sd larger than 0. data(golub. (b) The gene with standard deviation larger than 0.golub. data(golub. (a) Computation of mean gene expression values.agrep("^oncogene". package="multtest") rowindex <. (b) By the script below the "Cellular oncogene c-fos is found. Oncogenes in Golub data.5 is 1498.3] [1] "U43901_rna1_s_at" "M13934_cds2_at" (d) Give their biological names.gnames[o[1:3]. > golub.gnames[.order(meangol. 5.1. Computations on gene standard deviations of the Golub data.golub[sdgol>0. (c) sum(sdgol>0.mean) (b) To order the data frame use o <.] (c) Give the names of the three genes with the largest mean expression value.5 can be selected by golubsd <. (a) The standard deviation per gene can be computed by sdgol <apply(golub.decreasing=TRUE) and golub[o.2]) oncogol <. (a) length(agrep("^oncogene".].2] [1] "37 kD laminin receptor precursor/p40 ribosome associated protein [2] "RPS14 gene (ribosomal protein S14) extracted from Human ribosoma [3] "GAPD Glyceraldehyde-3-phosphate dehydrogenase" 4. ANSWERS TO EXERCISES 3.apply(golub.gnames[o[1:3].2])) gives 42.] .golub[rowindex. package = "multtest") meangol <. Be aware of the correct column separation.c("probe ID".15995 Answers to exercises of Chapter 2: Descriptive Statistics and Data Display .gnames[o[1:3].fac=="ALL"].2)] colnames(x) <.4).fac=="AML"].decreasing=TRUE) > meanB1[o[1:3]] AFFX-hum_alu_at 31962_at 31957_r_at 13.gol.decreasing=TRUE) > oncogolub.2] [1] "PIM1 Pim-1 oncogene" "JUNB Jun B proto-oncogene" [3] "Proto-oncogene BCL3 gene" (d) Writing results to a csv ﬁle.golub. (b) gl(5.table(x. 7.file="goluboutcsv") write.apply(oncogol[."AML")) meangol <.order(meangol.1.1. x <.order(meanB1.file="goluboutnorowname".apply(oncogol[.gnames <. (c) gl(3.1.decreasing=TRUE) > oncogolub.41648 13. Gene means for B1 patients. library(ALL).order(meangol.3).c(3.factor(golub.] gol.16671 13.cl.apply(exprs(ALL[.ALLBT=="B1"]). Constructing a factor.mean) o <.gnames[o[1:10].221 oncogolub.5).fac <. data(ALL) meanB1 <.gol.names=FALSE) 6.levels=0:1.gnames[o[1:3]. (a) gl(2.gnames[rowindex."gene name") write. mean) o <.2] [1] "PIM1 Pim-1 oncogene" "JUNB Jun B proto-oncogene" [3] "Proto-oncogene BCL3 gene" (c) meangol <.mean) o <.row.csv(x.oncogolub. labels= c("ALL".

fac=="ALL"]. (c) The outlier increased the mean as well as the standard deviation. package="multtest") gol.gol.155108 8. data(golub.4 and dramatically increased the standard deviation 12.fac=="ALL"]) to observe that nearly all values of 66 are on the line.function(x) mean(x)/sd(x)) o <. (b) Use qqnorm(golub[i.1.]~gol. Eﬀect size. The mean (-1.gol. Illustration of mean and standard deviation. but those of row 790 are not. (a) Use x<.7905694.factor(golub. HTF10)" [3] "HnRNP-E2 mRNA" [4] "54 kDa protein mRNA" [5] "Immunophilin homolog ARA9 mRNA" .790.28137) due to outliers on the right hand side. Hypothesis: The expression values of 66 are normally distributed.638308 9.2. ANSWERS TO EXERCISES 1.5.order(efs.gol.66 or i <. Take i <.fac) to observe that 790 has three outliers and 66 has no.apply(golub[. The diﬀerences are smaller.fac=="ALL"]) and median(golub[i.gol.gol."AML")) efs <.1.5.fac <.695353 > golub.2] [1] "YWHAZ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activa [2] "ZNF91 Zinc finger protein 91 (HPF7. (a) Use boxplot(golub[i.levels=0:1.64615. because the mean is eleven times larger than the standard deviation.23023.decreasing=TRUE) > efs[o[1:5]] [1] 11.222 APPENDIX A.138128 10.cl.fac=="ALL"]). (b) Now the mean is 7.174024) is larger than the median (-1.182503 and the median 1.c(1.gnames[o[1:5]. (a) The size 11 is large. Comparing two genes. (c) Use mean(golub[i.fac=="ALL") and qqline(golub[i.3) and mean(x) and sd(x) to obtain the mean is 2 and the standard deviation is 0.2. 2.954115 8. where as for 790 the three outliers are way of the normality line. For the gen in row 66 the mean is 1. labels= c("ALL". 3.

function(x) median(x)/mad(x)) o <. locator() x11() x <.2] [1] "COX6B gene (COXG) extracted from Human DNA from overlapping chromosome 1 F25451. vertical = TRUE) stripchart(golub[1042.vertical = TRUE) stripchart(golub[1042. pch="*".4)) . data(golub.cl.fac <.levels=0:1.1.] ~ gol.col=c("red".gol.27698 13.method="jitter".fac.xlim=c(0. alpha su isoform 1. genomic sequence" [2] "AFFX-HSAC07/X00351_M_at (endogenous control)" [3] "ATP5A1 ATP synthase.levels=0:1.cl.apply(golub[.fac.gnames[o[1:5]. The answers in the script below. "blue").method="jitter".91608 > golub.57425 13. cardiac muscle" [4] "ATP SYNTHASE GAMMA CHAIN.method="jitter". mitochondrial F1 complex.] ~ gol.decreasing=TRUE) > refs[o[1:5]] [1] 14.] ~ gol.data(golub.223 (b) The robust variant can be deﬁned by dividing the median by the MAD.factor(golub.order(refs. labels= c("ALL"."AML")) boxplot(x. This gives other best genes indicating that the some genes may have outliers that inﬂuence the outcome.fac=="ALL"].method="jitter") stripchart(golub[1042. Plotting gene expressions "CCND3 Cyclin D3"."AML")) stripchart(golub[1042. refs <.factor(golub.51217 13.fac <. package = "multtest") gol. and R31076 containing COX6B and UPKA. An alternative would be to divide the median by the IQR. "blue"). H+ transporting. Box-and-Whiskers plot of "CCND3 Cyclin D3". labels= c("ALL". MITOCHONDRIAL PRECURSOR" [5] "YWHAZ Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation pro zeta polypeptide" 4.fac.fac.vertical = TRUE) title("CCND3 Cyclin D3 expression value for ALL and AMl patients") 5.14419 12.] ~ gol.. package = "multtest") gol.col=c("red".

24.06922).24.5.1."lower wisker") arrows(2.93).59).1.fac <.27.93. do.mad)) [1] 0.0.stats(x.file="BoxplotWithExplanation.5.1) .text(2.golub[rowindex.1.1. coef = 1.mean) personmedian <.1.0.59.24.59. All persons have outliers near three. (a) The medians are all around zero.1.349)) [1] 0.24. The script below will do.15383. labels= c("ALL".text(2.out = TRUE) #finds value 6.1.2.1.text(2.2420185 7. 0.0.copy2eps(device=x11.2.27).5.0. the minimal values are all around minus 1.cl. (b) The means are very close to zero.5.2.2."third quartile") arrows(2. ANSWERS TO EXERCISES arrows(2.1.1.1. (a) Note that we need the transpose operator t to change rows into columns.5.2.text(2.1. data(golub.] .median) (c) The data seem preprocessed to have standard deviation equal to one."Outlier") arrows(2.27.5.2.text(2.5.agrep("^oncogene".] oncogolub.function(x) IQR(x)/1.eps") boxplot.. (1999) data.17).0.1.1.levels=0:1.2.17. the inter quartile range diﬀer only slightly. (1999) data.text(2."AML")) rowindex <.79.apply(golub.1."Median") arrows(2.93.apply(golub.sd)) [1] 0.79).2. > range(apply(golub.factor(golub. The medians are all between (−0.0. Box-and-wiskers plot of persons of Golub et al.9590346 1.2. so these are also close to zero."first quartile") arrows(2.17.224 APPENDIX A.79.golub.1. do. personmean <.conf = TRUE.9999988 1.gnames <. package="multtest") gol.2]) oncogol <.golub.gnames[.2.1. The re-scaled IQR and MAD have slightly larger range."upper wisker") dev.9599036 1.24.1.2.gnames[rowindex.3361527 > range(apply(golub.24.1. Oncogenes of Golub et al.0000011 > range(apply(golub.1.5.

225 row.gol.frame(t(oncogol[.330984 3.median)) [1] -1.1.3] boxplot(data.function(x) IQR(x)/1.gol. but others certainly have. Descriptive statistics for the ALL gene expression values of the Golub et al.mean)) [1] -1.mad)) .1.gnames[.1)) 8. Also. The range of the mean and of the median are similar. (1999) data.fac=="ALL"].fac=="ALL"]))) title("Box and wiskers plot for oncogenes of ALL patients ") boxplot(data.fac=="ALL"].fac=="ALL"].0381309 > range(apply(golub[. some do not have outliers.fac=="ALL"]))) (b) The plot gives a nice overview of the distributions of the gene expressions values of the onco gene separately for the ALL and the AML patients. > range(apply(golub[.names(oncogol) <. but those for AML are larger than zero. (a) The ranges indicate strong diﬀerence in means.1153113 2.fac=="ALL"].349)) [1] 0.gol. but others not. some have a small inter quartile ranges.1336206 2. Some gene show distinct distributions between patient groups. > range(apply(golub[. while for others this is large.gol.oncogolub. A similar statement holds for outliers.1)) boxplot(data. For instance.7769867 > range(apply(golub[.gol.frame(t(oncogol[.gol.sd)) [1] 0.frame(t(oncogol[. The bulk of the data seems symmetric. the sixth has ALL expressions around zero. Several genes behave similarly for ALL and AML. par(mfrow=c(2.fac=="AML"]))) title("Box and wiskers plot for oncogenes of AML patients ") par(mfrow=c(1.1.gol.gol.278551 > range(apply(golub[.1.1. Some are clearly distributed around zero.fac=="ALL"].35455 (b) The range of the standard deviation is somewhat smaller than of the re-scaled IQR and MAD.36832 3.

91993.02) = 0.644854. (d) The quantiles x0.226 APPENDIX A.975 = 13. T-distribution.95 = 1.4 *0. (a) P (X < 12) = 0.8830403. Normal.025 = 6. P (20 ≤ X) = 0. (a) P (1.6) (e) x0. Standard Normal. .8830403.5557756.975 = 32.025 = 17. (d) P (0 < Z < 1. (b) P (X > 8) = 0. (b) P (Z < 1.1034. z0.0746237.025 = −1. 2.1046692.05 = −1.999975. var(X) = 3.6440823. (b) P (T6 > 2) = 0. and z0.64 < Z < −1.975 = 1.6 < Z < 2.83856.9500.8413. x0.4750.9495.96) = 0.8413.3) = 0.5 = 10.1056649 2.959964. P (X ≤ 24) = 0. (f) z0.2917.9656744 Answers to exercises of Chapter 3: Important Distributions 1. (a) P (T6 < 1) = 0. (b) P (20 ≤ X ≤ 30) = 0.794733 Use: sqrt(60 * 0. (e) P (−1.96) = 0. and P (20 ≤ XandX ≥ 10) = 0. x0. 3.8220412.04621316.959964. z0.5 = 0.64) = 0. (c) P (9 < X < 10. and x0.5 = 24. 5) = 0. (c) P (−1. and x0.080072. (c) P (−1 < T6 < 1) = 0. (d) E(X) = 24. (c) P (20 ≤ XorX ≥ 40) = 0. 4. ANSWERS TO EXERCISES [1] 0.04408. Binomial (a) P (X = 24) = 0.644854. z0.96 < Z < 1. and P (X ≥ 30) = 0.

04939.7) − pbinom(9.025 = −2. (d) The quantiles f0. F distribution. (c) P (X > 10) = 1 − P (X ≤ 10) = 1 − pbinom(10. 20.7 * 0. 10 (b) P (χ2 > 4) = 0.0. 0.5 = 9.025 = 3.6.8) =pnorm(2.025 = 0. (a) P (X = 14) = dbinom(14.054510.9520381.4)=0.pnorm(1. .0.0. 0. 7.8.0) =pnorm(2. (a) P (F8.8792198.975 = 20.4)=0.5 > 4) = 0. (b) P (F8.2. (e) t0. (b) P (X ≤ 14) = pbinom(14. 0.6.4.6.975 = 2. and t0.5 = 1.4931282.9075737.1.1845646.07169537. and f0.1586553. 20. 8.48318.4) .4 ≤ X ≤ 0.191639.7) = 0.947347.7453474.0.446912. t0. f0.5 = 0. g0.7 = 14.6.246973. (d) P (10 ≤ X ≤ 15) = P (X ≤ 15)−P (X ≤ 9) = pbinom(15.2.6.1.227 (d) P (−2 < T6 < −2) = 0.pnorm(0. 10 (d) The quantiles g0.7) = 0. Zyxin.01857594. (e) 20 · 0. 20. 5. Chi-squared distribution. 10 (c) P (1 < χ2 < 6) = 0. (a) P (χ2 < 3) = 0.3)=2. 0.446912.2 ≤ X ≤ 2.7) = 0. (f) sqrt(20* 0.4)=0.2075862.5 < 3) = 0.4) .6826895. 20.975 = 6. (b) P (1.2) =pnorm(1. and g0.5836292.0.1. (c) P (2.1. MicroRNA. 0. (a) P (X ≤ 1. (c) P (1 < F8. 6.341818.5 < 6) = 0.0. 20.7) = 0.1.9544997.757172.

55. data(golub.gol.5) 10.levels=0:1.1.fac <. Extreme value investigation. sdall <.col = "red") Answers exercise chapter 4: Estimation and Inference 1. sd) sdratio <.5 are extremely large.apply(golub[. package="multtest") gol.228 APPENDIX A.decreasing=TRUE) tval[o[1:3]] golub.add=TRUE.383986. 9. (a) The code .ylim=c(0.8160144.col = "blue") curve(dnorm.025 =qnorm(0. (a) The tree larges t-value 57.double().5.sdall/sdaml sum( sdratio > 0.4) gives mean(x) = 1.4022082.gnames[. The blue line (extreme value) ﬁts to the black line (density of generated extreme data) much better than the red line (normal distribution). n <.5*(log(log(n))+log(4*pi))*(2*log(n))^(-1/2) bn <. Both are close to the values in the population.0.rnorm(1000.range(density(e)$x).apply(golub[.golub.975 = 2.10000 # Serfling p.gol.2]) to ﬁnd 808. (1999) data. and 47.fac=="AML"].gnames[o[1:3]. sd) sdaml <.2] (b) The scrip below gives 2185 ratios between 0.0.0.5 and 1. x0.90 for (i in 1:1000) e[i] <.order(tval.025.8.factor(golub. Gene CD33. Use agrep("^CD33". (e) x <.1.apply(golub[.2. ANSWERS TO EXERCISES (d) x0.6.6.1.gol.5 & sdratio < 1. Similarly.(max(rnorm(n))-an)/bn plot(density(e).4)=0.function(x) sqrt(27) * mean(x) o <.608401 and sd(x)=0.1.5)) f<-function(x){exp(-x)*exp(-exp(-x))} curve(f. an <.fac=="ALL"]."AML")) tval <.(2*log(n))^(-1/2) e <. labels= c("ALL".sqrt(2*log(n)) .0. Some computations on Golub et al.cl.1.fac=="ALL"].add=TRUE. Use i <. (b) Use chisq. t = -7.fac) gives p-value = 0.318e-07.cl.data(golub) gol.] ~ gol.test(as. so the null hypothesis of no outliers is rejected. Hence. Zyxin. so that normality is rejected. so that equality of means is rejected.test(golub[i. Note that the p-value from Grubbs test of the ALL expression values is 0.592 and changing ALL into AML gives p-value = 0. 2. var.1788.test(golub[i. var.test(golub[i.test(golub[i.2"))))$Freq) to ﬁnd p-value < 2.] ~ gol.2583.fac <.fac=="ALL"]) gives p-value = 1.gol.fac. Gene MYBL2 V-myb avian myeloblastosis viral oncogene homolog-like 2. so equality of means is rejected. (b) var. (a) shapiro. .] ~ gol. (b) t.equal = TRUE) gives p-value = 1.229 library(multtest).00519.923e-05. (a) Searching NCBI UniGene on zyxin gives BC002323.] ~ gol.factor(golub.fac) to observe from the boxplot that one is quite certain that the null-hypothesis of no experimental eﬀect holds. 4."AML")) shapiro.1095 so equality of variances is accepted.2.frame(table(read.] ~ gol.8597.3026 and is quite large. so that the null-hypothesis of equal frequencies is rejected.GenBank(c("BC002323.fac) gives p-value = 7.773e-09.data. so that the null hypothesis of equal means is accepted. Take i <.9813 is quite extreme.fac.fac=="ALL"]) gives p-value = 0.levels=0:1.equal = TRUE) gives p-value = 0. HOXA9.test(golub[i. labels= c("ALL". (a) Use boxplot(golub[i.test(golub[i.1391. for normality is accepted. 3.2e-16. (d) Yes. Its t-value equals -4. Nevertheless the Welch twosample T -test is also rejects the null-hypothesis of equal means.gol. (c) t. (b) wilcox.

3/16.as. x <.test(x=c(930. function(x) t. ANSWERS TO EXERCISES (c) We download and store the frequencies of the sequences in x and y.cl.levels=0:1.0277.2379 8.p=c(9/16. 290.factor(golub. From the output below the null hypothesis that the probabilities are as speciﬁed is accepted.index<-golub[index. data(golub) gol. df = 3.] pt. p-value = 0. function(x) t.1"))))$Freq y <. p-value = 0.GenBank(c("X94991.gnames[order(ptg)[1:10].test(x ~ gol.apply(golub.gnames[index.as. .2] 7. Next the empirical probabilities from y are use to predict the frequencies from y. Comparing two genes.2276. > chisq.9988 5.fac)$p.index[order(pt.2"))))$Freq >chisq. gol. 1. ptg <. 1.test(x.apply(golub.290.index<-golub.index<-pt[index] golub.2]) golub.1/16)) Chi-squared test for given probabilities data: c(930."AML")) pt <.2] 6.frame(table(read.frame(table(read.gnames[.fac.] golub. 330.GenBank(c("BC002323. Genetic Model.90).golub.330.value) golub.fac <.data.gnames.test(x ~ alternative = c("greater"))$p.3/16.data.gnames. library(multtest). Antigenes. Gene selection. p=y/sum(y)) Chi-squared test for given probabilities data: x X-squared = 0. labels= c("ALL".value) index <-agrep("^antigen". 90) X-squared = 4.230 APPENDIX A.index)[1:length(index)]. df = 3.

apply(golub. labels= c("ALL".apply(golub[.apply(golub.27598 10. gol.gnames[o[1:10]. 1.factor(golub.sd(all790) IQR(all66)/1.shapiro. (1999) data."AML")) allsh <.test(x)$p.test(x ~ gol. Normality tests.fac=="AML"].mad(all790) shapiro.fac=="ALL"] boxplot(all66.05 & allsh>0.fac=="ALL"].test(x)$p.gol.fac <.gol.value) > 100 * sum(allsh>0. library(multtest).05)/length(amlsh) [1] 78.fac)$p.fac)$p.value) o <.2] [1] "Zyxin" [2] "FAH Fumarylacetoacetate" [3] "APLP2 Amyloid beta (A4) precursor-like protein 2" [4] "LYN V-yes-1 Yamaguchi sarcoma viral related oncogene homolog" [5] "CST3 Cystatin C (amyloid angiopathy and cerebral hemorrhage)" .fac=="ALL"] all790 <.factor(golub.levels=0:1. 1. function(x) t.IQR(all790)/1.value) amlsh <. labels= c("ALL". function(x) shapiro.data(golub) gol.mean(all790) mad(all66).test(x ~ gol. 1.5644 > 100 * sum(allsh>0.golub[66.05)/length(allsh) [1] 58.27598 > 100 * sum(amlsh>0. package = "multtest").test(all790) 9.test(all66).order(pt.mean(all790) median(all66). (a) data(golub.cl."AML")) pt <.decreasing=FALSE) > golub.231 all66 <.cl.levels=0:1.gol. function(x) wilcox. 1.apply(golub[.05)/length(allsh) [1] 58. Two-sample tests on gene expression values of the Golub et al.golub[790. function(x) shapiro.349 mean(all66).median(all790) sd(all66).fac <.value) pw <.all790) mean(all66).gol.349 .

232 [6] [7] [8] [9] [10] APPENDIX A.factor(golub.golub[1042.gol.05))=1.golub[1042. (a) data(golub...v)* sqrt(var(x)/n + var(y)/m) mean(x) .v)* sqrt(var(x)/n + var(y)/m) .05)=5.order(pw. (c) sum(dbinom(6:1000. (a) n = 1000.levels=0:1.2] [1] "FAH Fumarylacetoacetate" [2] "Zyxin" [3] "CST3 Cystatin C (amyloid angiopathy and cerebral hemorrhage)" [4] "ELA2 Elastatse 2.decreasing=FALSE) > golub.gol.1000.(mean(x)-mean(y))/sqrt(var(x)/n + var(y)/m) v <."AML")) x <. Biological hypotheses.1000.8 · 10−14 . labels= c("ALL".length(y) t <.length(x) y <..fac=="AML"] m <.025.fac <.mean(y) + qt(0.mean(y) + qt(0.05))= 8. GAMMA SUBUNIT" (b) > o <.v) mean(x) .(var(x)/n + var(y)/m)^2/( (var(x)/n)^2/(n-1) + (var(y)/m)^2/(m-1 2*pt(-abs(t). neutrophil" [5] "TCF3 Transcription factor 3 (E2A immunoglobulin enhancer bindin [6] "Macmarcks" [7] "LYN V-yes-1 Yamaguchi sarcoma viral related oncogene homolog" [8] "CD33 CD33 antigen (differentiation antigen)" [9] "VIL2 Villin 2 (ezrin)" [10] "APLP2 Amyloid beta (A4) precursor-like protein 2" 11.05 so np = 50 (b) pbinom(9. Programming some tests.1000. p = 0.fac=="ALL"] n <. ANSWERS TO EXERCISES "X-LINKED HELICASE II" "RB1 Retinoblastoma 1 (including osteosarcoma)" "TOP2B Topoisomerase (DNA) II beta (180kD)" "TCRA T cell receptor alpha-chain" "T-COMPLEX PROTEIN 1.package="multtest") gol. 12.975.gnames[o[1:10].24 · 10−13 . (d) sum(dbinom(2:8.cl.

"B1"."B2".fac=="ALL"] y <."B4")] > table(ALLB$BT) B B1 B2 B3 B4 5 19 36 23 12 T T1 T2 T3 T4 0 0 0 0 0 psw <. Analysis of gene expressions of B-cell ALL patients.golub[1042.05) [1] 6262 2.gol. 1. function(x) anova(lm(x ~ ALLB$BT))$Pr[1]) > featureNames(ALLB)[panova<0.] > sum(rank(z)[1:27]) .test(residuals(lm(x ~ ALLB$BT))) library(lmtest) pbp <-apply(exprs(ALLB).05 & pbp > 0."B3".golub[1042. data(ALL) ALLB <.gol.000001] [1] "1125_s_at" "1126_s_at" "1134_at" "1389_at" "1500_at" .fac=="AML"] w <.5*27*(27+1) [1] 284 (c) x <.0.ALL$BT %in% c("B". function(x) as.studentize = FALSE)$p.apply(exprs(ALLB).ALL[.w + sum(x[i]>y) > w [1] 284 Answers to exercises of Chapter 5 Linear Models 1.apply(exprs(ALLB).233 (b) z <.value)) > sum(psw > 0.0 for (i in 1:27) w <.05) [1] 6847 > sum(pbp > 0.05) [1] 10057 > sum(psw > 0. 1.numeric(bptest(lm(x ~ ALLB$BT). function(x) shapiro. library(ALL). > panova <. Further analysis of gene expressions of B-cell ALL patients.golub[1042. 1. apply(exprs(ALLB).001 > table(panovasmall.348192e-09 7.145502e-09 4.873245e-10 1. Finding the ten best best genes among gene expressions of B-cell ALL patients.719456e-06 npanova <.748615 36873_at 1866_g_at 2.997065e-08 38555_at 1866_g_at 40155_at 191 1.va > featureNames(ALLB)[pkw<0.402379e-08 3.test(x ~ ALLB$BT)$p. There are only 38 signiﬁcant gene expressions from Kruskal-Wallis which are non-signiﬁcant according to ANOVA.891702e-14 40763_at 37809_at 5. function(x) kruskal. ANSWERS TO EXERCISES [6] "1866_g_at" "1914_at" "205_g_at" "31472_s_at" "31615_i_at" [11] "31616_r_at" "33358_at" "35614_at" "35991_at" "36873_at" [16] "37809_at" "37902_at" "38032_at" "38555_at" "39716_at" [21] "40155_at" "40268_at" "40493_at" "40661_at" "40763_at" [26] "41071_at" "41139_at" "41448_at" "873_at" > pkw <.000001] [1] "1389_at" "1866_g_at" "38555_at" "40155_at" "40268_at" > panovasmall <.001 > pkwsmall <.764046e-08 1125_s_at 40662_g_at 1.346907e-06 1.335279e-07 6.pkwsmall) pkwsmall panovasmall FALSE TRUE FALSE 12172 38 TRUE 124 291 There are 124 signiﬁcant gene expressions from ANOVA which are not signiﬁcant on Kruskal-Wallis.074525 38032_at 40661_at 1.117406e-09 1.123068e-07 2.256410e-09 2.names(sort(panova)[1:10]) npkw <. > sort(panova)[1:10] 1914_at 1389_at 1. 1.panova < 0.595926e-07 1.names(sort(pkw)[1:10]) .475170e-06 1.234 APPENDIX A.pkw < 0.466523e-14 5. 3.155457e-08 > sort(pkw)[1:10] 1389_at 40268_at 2. The tests agree on the majority of gene expressions.384281e-06 38555_at 33358_at 40268_at 3971 4. 05 · 10.apply(data.1.10000 data <.nrow = 10000. A matrix with diﬀerences between three groups of gene expression values. ncol = 3)) a <.matrix(rnorm(n*3.3) panova <. which is quite close to the observed.matrix(rnorm(90000). function(x) anova(lm(x ~ a))$Pr[1]) > sum(panova<0.3) > panova <. A simulation study for ANOVA.sigma). ncol = 3). 1.1. Answers to exercises of Chapter 6: Micro Array Analysis. For the Kruskal-Wallis test there are 1143 true positives and 8857 false negatives. ncol = 9) > a <. function(x) anova(lm(x ~ a))$Pr[1]) > sum(panova<0. "38555_at" "40268_at" "1866_g_at" .2. matrix(rnorm(n*3.gl(3. Gene ﬁltering on normality per group of B-cell ALL patients.gl(3.npkw) [1] "1914_at" "1389_at" 4. 1.05) [1] 1143 Thus the number of true positives from ANOVA is 3757 and the number of false negatives is 6243. function(x) kruskal.sigma). 000 = 500.apply(data. 1. 1.sigma). n <. The expected number is α · n = 0.235 > intersect(npanova.ncol=3). > x <.apply(x.05) [1] 3757 > pkw <. sigma <.05) [1] 514 The number of false positives is 514. This can be impoved by increasing the number of gene expressions per group.test(x ~ a)$p.0.cbind(matrix(rnorm(n*3.value) > sum(pkw<0.

ma <. cont.function(x) (shapiro."B2". coef=2.handler()) saveHTML(anntable.ALL[.ALL$BT %in% c("B1". filterfun(f1)) sel3 <."sel4") vc <. "hgu95av2".vennCounts(x.B4-B3.genefilter(exprs(ALL[.test(x)$p."B4")] design.library(hgu95av2.makeContrasts(B2-B1.c("sel2". "ALLB1234.ma) <.levels=factor(ALLB$BT)) fit <.ncol = 3. etc. Finding a row number: grep("1389_at".sel3.contrasts.names(exprs(ALL))).eBayes(fit1) topTable(fit1."B4")] f1 <. filterfun(f1)) sel4 <.aafTableAnn(as.ALL[. Remission (genezing) from acute lymphocytic leukemia (ALL).B3-B2.3. library("ALL"). package = "ALL") ALLB <.ALLB$BT=="B3"]).genefilter(exprs(ALL[.html".value > 0. 4.ma) fit1 <."B2". number=20.sel1 & sel2 & sel3 & sel4 library(limma) x <.method="fdr") anntable <.adjust.ma <.05) sel1 <. . 2. coef=2. aaf.ALLB$BT=="B1"]).ma) fit1 <. ANSWERS TO EXERCISES library("genefilter") data(ALL."B3". include="both") vennDiagram(vc) 137 pass filter 2 but not the other 510 pass filter 2 and 3 but not 4 1019 pas filter 2 and 4 but not 3 5598 pass filter 2."sel3".genefilter(exprs(ALL[."B4") cont. design. filterfun(f1)) sel2 <.library("annaffy")."B3".5. filterfun(f1)) selected <.row."B3".2.method="fdr") tab <.lmFit(ALLB.sel4)).integer(c(sel2. library("limma"). title = "B-cell ALL of stage 1.236 APPENDIX A.matrix(as.4" 3.character(tab$ID).genefilter(exprs(ALL[.model.ALLB$BT=="B2"]). 3 and 4.db) data(ALL) ALLB <.fit(fit. adjust.c("B1".matrix(~0 + factor(ALLB$BT)) colnames(design."B2".ALL$BT %in% c("B1".ALLB$BT=="B4"]).topTable(fit1. Analysis of gene expressions of B-cell ALL patients using Limma.byrow=FALSE) colnames(x) <.

mget(names. data(ALL) ALLCRREF <.featureNames(ALLrem)[pano<.unlistednames) [1] 12 21 > length(unique(unlistednames)) [1] 36 5.names=F) > grep("p53".0001] [1] "1472_g_at" "1473_s_at" "1475_s_at" "1863_s_at" "34098_f_at" "36574_at" library("hgu95av2.mget(names.0001] genenames <.001) > sum(pano<0.1.001] ALLremsel<.db") affynames <.value) sum(pano<0.as.test(x ~ remfac)$p."REF"))] pano <.apply(exprs(ALLCRREF).value) > sum(pano<0.] symb <.function(x) t.unlist(listofgenenames[names].which(pData(ALL)$remission %in% c("CR".which(ALL$CR %in% c("CR".ALL[.test(x ~ ALLCRREF$CR)$p."REF")) ALLrem <.list(hgu95av2GENENAME) unlistednames <. data(ALL) table(pData(ALL)$remission) remis <.1.hgu95av2GENENAME) listofgenenames <.remis] remfac <-factor(pData(ALLrem)$remission) pano <.237 library(ALL). library(ALL).ALL[.ALLrem[names.featureNames(ALLCRREF)[pano<.0001) [1] 11 > featureNames(ALLCRREF)[pano<.apply(exprs(ALLrem).use.mget(affynames.function(x) t. env = hgu95av2SYMBOL) genenames <. env = hgu95av2GENENAME) .db) names <.001) [1] 45 library(hgu95av2. Remission achieved. 11. filterfun(f1)) sel3 <.2.function(x) (shapiro.mget(affytot.915642 sample estimates: odds ratio 14."T3"))] library(genefilter) f1 <.002047 alternative hypothesis: true odds ratio is not equal to 1 95 percent confidence interval: 2.239. Gene ﬁltering of ALL data.genenames) [1] 1 2 3 affytot <.which(ALL$BT %in% c("T2".ALLT23$BT=="T3"]).byrow=TRUE) > fisher.genefilter(exprs(ALLT23). filterfun(f2)) > sum(sel1 & sel2 & sel3) .05) sel1 <.value < 0.test(x ~ ALLT23$BT)$p.test(dat) Fisher’s Exact Test for Count Data data: dat p-value = 0.3).genenamestot)) [1] 239 > length(genenamestot) [1] 12625 > dat <.test(x)$p.05) f2 <. env = hgu95av2GENENAME) > length(grep("oncogene".39959 6.matrix(c(12625. filterfun(f1)) sel2 <.ALL[.genefilter(exprs(ALLT23[. library("ALL") data("ALL") table(ALL$BT) ALLT23 <.238 APPENDIX A.562237 54.genefilter(exprs(ALLT23[.unique(featureNames(ALLCRREF)) genenamestot <.value > 0. ANSWERS TO EXERCISES > grep("oncogene".function(x) (t.ALLT23$BT=="T2"]).

nrmissing pval486[pval486==0]<-pval486sel .test(x ~ eset486sel$cell pval486 <. coef=2."B2".makeContrasts(B2-B1.db).7248509 5.fit(fit.258491 5.ma) <.083524 2.model.0001825464 8.ma <.132884 6776 36711_at -2. 1.946078e-08 0. library(limma).8470235 4.ma) fit1 <.961231 4. Stages of B-cell ALL in the ALL data.5.0000325578 10.05) [1] 4328 8.lmFit(allB.log2=T) nrmissing <.B3-B2. design.Val B 6048 35991_at 0.891823e-08 0.624128 2. eset486 <.apply(exprs(eset486). 1.GDS2eSet(gds486.factor(allB$BT) cont."B2". function(x) sum(is. cont.276579e-07 0.matrix(~ 0 + facB123) colnames(design. function(x) t.0003054110 7. library(hgu95av2. library("ALL") library("limma"). levels=facB123) design.144598 6. library(GEOquery)."B4"))] facB123 <.na(x)) ) eset486sel <.] pval486sel <.576108 -5.ma) fit1 <.contrasts.Value adj.getGEO("GDS486").eset486[nrmissing<1.c("B1".0003054110 7.P.742783 1."B3". allB <.eBayes(fit1) > topTable(fit1.263298 > sum(fit1$p.ma <.3664712 7.0002081474 8. Analysis of public micro array data.B4-B3.which(ALL$BT %in% c("B1".217570 6.187487e-07 0.ALL[.value<0.5707770 7.method="BH") ID logFC AveExpr t P.842989 3909 33873_at 0.5964481 4.625253 5668 35614_at 1."B3".apply(exprs(eset486sel).329631 7978 37902_at 0.663477 5.578836e-09 0.adjust.library(annaffy) gds486 <.do.239 [1] 905 > sum(sel1 & sel2) [1] 9388 > sum(sel3) [1] 1204 7.759565 1."B4") fit <.

eset711 <.order(sumpval.apply(exprs(eset2126sel). 9. library(ALL) data(ALL.html") # p53 plays a role. function(x) sum(is.get(genenames[i]. env = hgu95av2SYMBOL) > symb [1] "GADD45A" "DUSP4" [16] "TKT" "NFKB1" "OAS1" "SLC7A5" "STAT1" "CXCL2" "STAT1" "DLG5" "AKR1C3" "PSMB9" library("KEGG").na(x)) ) eset711sel <. 1.do."aap" for (i in 1:20) symb[i] <. function(x) anova(lm(x ~ eset pval2126 <.nrmissing pval711[pval711==0]<-pval711sel pval711[pval711>1]<-1 gds2126 <.nrmissing pval2126[pval2126==0]<-pval2126sel pval2126[pval2126>1]<-1 sumpval <.eset2126[nrmissing<1. Analysis of genes from a GO search.library("GO").getGEO("GDS711").] panova711sel <.do.apply(exprs(eset2126). aaf.apply(exprs(eset711sel).panova711sel pval711 <.library("annaffy") atab <. function(x) anova(lm(x ~ eset pval711sel <.240 APPENDIX A.getGEO("GDS2126"). ANSWERS TO EXERCISES pval486[pval486>1]<-1 gds711 <. file="ThreeExperiments. 1.na(x)) ) eset2126sel <. eset2126 <.apply(exprs(eset711).eset711[nrmissing<1. "hgu95av2".decreasing=FALSE) genenames <.package="ALL") . 1.GDS2eSet(gds2126.aafTableAnn(genenames.log2=T) nrmissing <.names(sumpval[o[1:20]]) symb <.] pval2126sel <.log2=T) nrmissing <.handler() ) saveHTML(atab.pval486 + pval711 + pval2126 o <. function(x) sum(is.GDS2eSet(gds711. 1.

rep(2. function(x) anova(lm(x ~ nab.ALL$mol.factor(facnr.fac))$Pr[1]) library("GO").05) [1] 86 > sum(panova[probes]<1) [1] 320 > sum(panova<0. 1.biol=="BCR/ABL") orderpat <.function(term) { GTL <."BCR/ABL".rep(3. labels= c("NEG"."NEG")] ALLPo <."BCR/ABL".which(ALLP$mol.fac <.10). library("hgu95av2") GOTerm2Tag <.c(neg.ALL[.which(ALLP$mol."ALL1/AF4".aal1.2.which(ALLP$mol.ALL$mol. value=TRUE)}) Gl <.86).biol=="NEG") aal1 <.320.c(rep(1. library("annotate").241 ALLP <.biol %in% c("ALL1/AF4". length) names(GTL[Gl>0]) } > GOTerm2Tag("protein-tyrosine kinase") [1] "GO:0004713" probes <.74).levels=1:3.eapply(GOTERM.hgu95av2GO2ALLPROBES$"GO:0004713" > sum(panova[probes]<0.byrow=TRUE)) Fisher’s Exact Test for Count Data .sapply(GTL.apply(exprs(ALLPo).aal1.bcr)] facnr <.ALLP[.05) [1] 2581 > sum(panova<1) [1] 12625 > fisher.c(neg. x@Term. 2581.ALL[."NEG")] neg <.bcr) ALLP <."BCR/ABL")) panova <.test(matrix(c(12625.biol %in% c("ALL1/AF4".37)) nab. function(x) {grep(term.biol=="ALL1/AF4") bcr <. 5% -1.cl[i.] <.07569310 -0.fac. data(golub.as.replace=TRUE)] cl <.]) } > quantile(boot.cl$centers[2. nstart = 10) table(cl$cluster.method="euclidian").1].gol.data.numeric(gol.cl. initial.679625 sample estimates: odds ratio 1.factor(golub.0. (1999) data. Cluster analysis on the ”Zyxin” expression values of the Golub et al.kmeans(dat. package="multtest") data <.kmeans(data.mean). 1. 2581.matrix(0. nstart = 10) boot.levels=0:1.242 APPENDIX A.fac <.data[sample(1:n. there are more significan Answers to exercises of Chapter 7: Cluster Analysis and Trees.nrow=nboot.019848 1. cl<.].025.star <.cl[. byrow = TRUE) p-value = 0."AML")) stripchart(golub[2124.314569 the odds ratio differs significantly from zero.star. 2.method="single")) initial <. ANSWERS TO EXERCISES data: matrix(c(12625.5% 97. 86).].fac)) plot(hclust(dist(clusdata. 320.fac.c(0.]) gol. labels= c("ALL". nboot<-1000 boot.matrix(tapply(golub[2124.gol.03222 alternative hypothesis: true odds ratio is not equal to 1 95 percent confidence interval: 1.03344292 . pch=as.length(data).]~gol.ncol = 2) for (i in 1:nboot){ dat.fac) n <. initial. ncol=1.c(cl$centers[1.975)) 2. nrow = 2.cl <.frame(golub[2124.

10.y[-21]).] <.7690824 0.fac) 3.y) which.] ~gol.cl[.025.9324625 # very similar to cor.genefinder(golub.731493 1. 1042. method = "euc".byrow=FALSE) for (i in 1:nboot){ dat.star)[2.data[sample(1:nrow(data).1]} > mean(boot.0.ncol = 1) data <. y <.cor[. library("genefilter").]. package = "multtest") x <.42e-12 alternative hypothesis: true correlation is not equal to 0 95 percent confidence interval: 0. package = "multtest") closeg <.975)) 2.gnames[closeg[[1]][[1]].6949.0. data(golub. MCM3.min(y) # the plot suggests the smallest y as the outlier > cor.cor) [1] 0.784468 2.test .matrix(c(x[-21].golub[2430.replace=TRUE).2] boxplot(golub[394.875043 # much larger than 0.nrow=nboot.cor(dat. df = 35.8725835 # very similar to cor. data(golub. scale = "none") golub.5% 0.golub[2289.2].9341905 # much smaller sample estimates: cor 0.] plot(x.star <.matrix(0.c(0.cor[i.c(0.5% 97.y[-21]) Pearson’s product-moment correlation data: x[-21] and y[-21] t = 10.cor <.test(x[-21].5% 0.243 > quantile(boot.025.1].test > quantile(boot.7755743 0.ncol=2. boot.975)) 2. p-value = 1.] boot. Close to CCND3 Cyclin D3.6376217 nboot <.5% 97.1000.

1.2]) plot(hclust(dist(golub[o1.9652965 2.replace=TRUE).5% 2.nrow(data) .exprs(ALLBsp).golub.levels=0:1.method="single")) o3 <.5% 97.5% .golub.c(0.ncol(data).2]) plot(hclust(dist(golub[o3.factor(golub.gnames[.data(golub). Principal Components Analysis on part of the ALL data. library(ALL). labels= c("ALL".5% 97.244 APPENDIX A. Cluster analysis on part of Golub data.].gnames[.5% 2.method="single")) 5.975))) 2.0.001.43550 66.] eigenvalues[i. library(multtest).081573 2.golub.grep("oncogene".] <.025.2]) plot(hclust(dist(golub[o2.5% 97. nboot<-1000 eigenvalues <. p <.p)) for (i in 1:nboot){dat.5% 63.data[sample(1:n.method="euclidian")."AML")) o1 <.fac <.gnames[.j].530350 2.grep("antigene". function(x) anova(lm(x ~ ALLB$BT))$Pr[1]) ALLBsp <.5805595 > eigen(cor(exprs(ALLBsp)))$values[1:5] [1] 65. gol.2016203 2.star))$values} > for (j in 1:p) print(quantile(eigenvalues[.cl."B2"."B3")] panova <.] > dim(exprs(ALLBsp)) [1] 499 78 > min(cor(exprs(ALLBsp))) [1] 0.method="euclidian"). n <.array(dim=c(nboot.575413 3.grep("receptor".ALL[.apply(exprs(ALLB).6040647 data <.ALL$BT %in% c("B1".77785 2.ALLB[panova<0.5% 97. data(ALL) ALLB <.].7556439 0.eigen(cor(dat.method="single")) o2 <.889933 2.method="euclidian"). ANSWERS TO EXERCISES 4.4781567 0.star <.]. ] -0.5% 97.5.cex=0.5.0.0.8164966 0.mingol) [1] 2124 > golub.5.-0.0.6/3 * 100 [1] 86.cor=TRUE). text(gol.pc.rp.245 0.8.5067404 0.5.1).5773503 -0.8.-0.fac) gol. Use recursive partitioning in rpart library(multtest).-0. Some correlation matrices.gol.7482680 2.5% 0. function(x) min(x)) sum(maxgol < mingol) > which.0.fac=="AML"].rp.nrow=2)) eigen(matrix(c(1.1). the first three are significant! biplot(princomp(data.1).-0.fac=="ALL"].5.rpart(gol. branch=0.] -0. Classiﬁcation tree of Golub data.8."AML")) maxgol <. 1.0.0.data(golub).5773503 -0.fac ~ golub[2124.].8.cl.levels=0:1.1.min(maxgol .0000000 [2.0. cp=0.8) 6.9942871 2.apply(golub[.0. eigen(matrix(c(1.1] [.1. digits=3.] ~gol. gol.8.66667 > eigen(matrix(c(1.fac <.5.8.-0.2] [.5773503 0.nrow=3)) eigen(matrix(c(1.rp <.7071068 Answers to exercises of Chapter 8: Classiﬁcation Methods.factor(golub.1). method="class".-0.5% 97.biplot=T.0. 1.nrow=3))$vectors [.5.-0.-0.8.0.gol.8.6475809 0.8.apply(golub[.8. 1.8.4082483 0.1). labels= c("ALL".nrow=3)) > 2.3] [1.1.0.n=TRUE) .001) plot(gol. use.0.8.8.7071068 [3.] [1] "4847" "Zyxin" "X95735_at" > boxplot(golub[2124. function(x) max(x)) mingol <.4082483 -0.gnames[2124.] -0.5% #Hence.expand=0.8.margin=0.

fac ~.method="class".groups) groups predicted 1 2 1 41 12 .clchanged) perf <.clchanged <.rp.frame(t(golub)). use.1).data(golub) golub.prediction(golub[1042.n=TRUE) gol. branch=0.]. plot(gol.2]) [1] 2058 gol.rpart(gol. method="class". Comparing Classiﬁcation Methods. cp=0.data.rp. golub. data.performance(pred. use. digits=3.50) simdata <.cl +1 pred <.100.1). digits=3. (c) Use auc as before.margin=0.rp <.gl(2.predict(rp. method="class".matrix(rnorm(100*4.type="class") table(predicted. text(gol.. ANSWERS TO EXERCISES grep("Gdf5". branch=0.rpart(gol.n=TRUE) 2.margin=0. text(gol. "spec") plot(perf) (b) The function is essentially the same.letters[1:4] groups <.predictors) rp<-rpart(groups ~ a + b + c + d.margin=0.rp <.1).gnames[.fac ~ golub[2058.data=simdata) predicted <.].frame(groups. (a) library(multtest).4) colnames(predictors) <.0. branch=0.n=TRUE) > table(predicted.2]) > grep("Gdf5". text(rp.golub.246 APPENDIX A.golub.rp. use.library(ROCR). digits=3. 3.gnames[.1). "sens".-golub.001) plot(gol. Sensitivity versus speciﬁcity.rp. cp=0.groups) plot(rp. library(rpart) predictors <.

n=TRUE) rpart. method="class".rp.remfac) remfac . type="class") > table(rpart. decay = 0.pred <. size = 5.ALL[.pred.predict(nnest. groups. branch=0. library(hgu95av2. text(all. type = "C-classification".featureNames(ALLrem)[pano<. respectively. probability=TRUE) > table(svmpred.value) names <.247 2 9 38 library(e1071) svmest <.svm(predictors.maxit = 500.data.1. library(ALL).ALLrem[names. 4..01. data(ALL) ALLrem <.rp. predictors. data=df.1). Max pred <.predict(all. groups) groups svmpred 1 2 1 31 25 2 19 25 library(nnet) nnest <.] data <. If we increase the number of predictors.nnet(groups ~ . library(rpart). then the misclassiﬁcation rate decreases.predict(svmest.rp. svm.which(pData(ALL)$remission %in% c("CR". kernel = "l svmpred <. data = simdata. Prediction of achieved remission.001] ALLremsel<.test(x ~ remfac)$p. groups) # prints confusion ma groups pred 1 2 1 45 10 2 5 40 The misclassiﬁcation rate of rpart.rp <.001) plot(all. data. use.margin=0.. 44/100. digits=3."REF"))] remfac <-factor(pData(ALLrem)$remission) pano <. type = "class") > table(pred. and 15/100.frame(t(exprs(ALLremsel))) all. 21/100.function(x) t. cp=0.db). and nnet is.rpart(remfac ~.apply(exprs(ALLrem). "im"."aac".values[[1]]) o <."lip"."pp")).fr/~torre/Recherche/Da downloads/ecoli/ecoli.rpart(ecolisel$ecclass ~ mcg + gvh + lip + aac + alm1 + alm2.] ecolisel$ecclass <.values."alm1". gol.pred CR REF CR 93 1 REF 6 14 > 7/(93+1+6+14) [1] 0."im".order(auc.factor(ecolisel$ecclass.apply(golub.header = TRUE) colnames(ecoli) <.2] 6.true <.levels=0:1. Classiﬁcation Tree for Ecoli.248 APPENDIX A.sep=". text(rpfit."gvh". use. ecoli <."36769_at"."mcg".cl.data"."1472_g_at".n=TRUE) title(main = "rpartfit ecoli classes cp im and pp") . library(ROCR). levels=c("cp". data(golub.labels= c("TRUE".ecoli[which(ecoli$ecclass %in% c("cp".1."pp")) library(rpart) rpfit <.margin=0."."chg". function(x) performance(prediction(x."854_at"). ANSWERS TO EXERCISES rpart.1)."auc")@y.gnames[o[1:25].read. Gene selection by area under the curve. branch=1. ecolisel<.grappa.06140351 > mget(c("1840_g_at".decreasing=TRUE) golub.true).values <. digits=3. package = "multtest") gol.table("http://www.univ-lille3.dat plot(rpfit."FALSE")) auc.factor(golub.c("SequenceName". env = hgu95av2GENE$‘1840_g_at‘ [1] NA $‘36769_at‘ [1] "retinoblastoma binding protein 5"$‘1472_g_at‘ [1] "v-myb myeloblastosis viral oncogene homolog (avian)" $‘854_at‘ [1] "B lymphoid tyrosine kinase" 5. wstep = par(mfrow=c(1. main = "Dot plot of equal random sequnces\nwsize = 3. seq2. file="ccnd3n. ws dotPlot(seq1. format="fasta".predict(rpfit.rep=TRUE. ws dotPlot(seq1. gvh and im > (1+2+7+4)/length(ecolisel$ecclass) [1] 0.sample(c("A".0. c2s) x1 <. 2.1)) par(mfrow=c(1.1.prob=c(0.2)) dotPlot(seq1.ecolisel$ecclass) #predictors are alm1."T"). seq1.fa".prob=c(0. ws . seq2.rep=TRUE.0. Dotplot of sequences.0. main = "Dot plot of equal random sequnces\nwsize = 1. seq1 <."sp=homo sapiens AND k=ccnd3@") ccnd3 <."T").100. width=80) ccnd3c2sn <."C".sapply(ccnd3.1)) par(mfrow=c(1."G".4.0. width=80) An alternative would be to use the write.05166052 Answers to exercises of Chapter 9: Analyzing Sequences 1. Writing to a FASTA ﬁle. choosebank("genbank")."C".2)) dotPlot(seq1.1.fa".XStringSet(x1.4.1)) seq2 <. library(seqinr) query("ccnd3hs".sample(c("A".0.XStringSet(x1. main = "Dot plot of different random sequences\nwsize = 3. type="class") table(predictedclass. seq1.0. main = "Dot plot of different random sequences\nwsize = 3."G". wstep = par(mfrow=c(1. format="fasta".4.2)) dotPlot(seq1.DNAStringSet(c2s(ccnd3[[1]])) write.4. file="ccnd3.dna function of the ape package. getSequence) x1 <.sapply(ccnd3hs$req.1)) par(mfrow=c(1.DNAStringSet(ccnd3c2sn) write. main = "Dot plot of different random sequences\nwsize = 1.249 predictedclass <. seq1.100.

1)) x <. library(seqinr) query("ccnd3hs".s2c("PAWHEAE") s <.matrix(data=NA. ccnd3prot[[8]]. s2c(z).] <. Local alignment.nrow=(length(y)+1).c2s(sample(rownames(BLOSUM50). getName) ccnd3prot <.c("RPLWVAPDGHIFLEAFSPVYK") z <. library(seqinr).s2c("HEAGAWGHEE").j]-d.10.c("".7.j-1]-d))} > max(F) [1] 28 4.1) for (i in 1:1000) { x <.1] <. d <.c("PLWISPSDGRIILESFSPLAE") choosebank("genbank").sapply(ccnd3hsreq.x) for (i in 2:(nrow(F))) for (j in 2:(ncol(F))) {F[i.j-1].ncol=(length(x)+1)) F[1. Probability of more extreme alignment score. getSequence) sapply(ccnd3hsreq. main = "Dot plot of two protein\n dotPlot(s2c(x).y). wstep 3.library(Biostrings).F[i.c2s(sample(rownames(BLOSUM50).j] <.sapply(ccnd3hs$req. main = "Dot plot of equal random sequnces\nwsi par(mfrow=c(1.250 APPENDIX A. s2c("EEEVFPLAMN"). seq1[100:1].c(""."sp=homo sapiens AND k=ccnd3@") ccnd3 <. y <. library(seqinr). ANSWERS TO EXERCISES dotPlot(seq1.0 rownames(F) <.8 F <. AAString(y).F[i-1. colnames(F) <.0 . F[.x].j-1]+s[i-1. substitut .BLOSUM50[y.data(BLOSUM50) randallscore <. main = "Dot plot of two protein\nwsize = 1. main = "Dot plot of two protei dotPlot(ccnd3prot[[7]].F[i-1. replace=TRUE)) randallscore[i] <. replace=TRUE)) y <. getTrans) dotPlot(ccnd3prot[[1]].library(Biostrings).pairwiseAlignment(AAString(x).c("RPLWVAPDGHIFLEAFSPVYK") y <.data(BLOSUM50) x <.max(c(0.c(1. 4507417 0."AC=AE017126 OR AC=BX548174 OR AC=BX548175") ccmpseq <.1079848 sample estimates: mean of x mean of y 0.gc[3:9]) Welch Two Sample t-test data: gc[1:2] and gc[3:9] t = -5. GC) > wilcox.getSequence) gc <."sp=homo sapiens AND k=ccnd3@") . gapExtension = -8.test(gc[1:2]. df = 1.251 gapOpening = 0. p-value = 0. Sequence equality.sapply(ccmpseq. \begin{verbatim} library(seqinr) choosebank("genbank") query("ccnd3hs".5075849 gc in the left group is lower. Prochlorococcus marinus. library(seqinr) choosebank("genbank") query("ccmp".test(gc[1:2].003 > plot(density(randallscore)) 5. 6.08649 alternative hypothesis: true difference in means is not equal to 0 95 percent confidence interval: -0. p-value = 0.3362065 0. the tests are not significant.05556 alternative hypothesis: true location shift is not equal to 0 > t. scoreOnly = TRUE) } > sum(randallscore>1)/1000 [1] 0.sapply(ccmp$req.gc[3:9]) Wilcoxon rank sum test data: gc[1:2] and gc[3:9] W = 0.8793.138.

AAString(z).getLength) > ccnd3prot <. there is only one difference! > which(!ccnd3prot[[1]]==ccnd3prot[[2]]) [1] 259 7.sapply(ccnd3hs$req.c("RPLWVAPDGHIFLEAFSPVYK") z <. ANSWERS TO EXERCISES sapply(ccnd3hs$req. AAString(y). W 3 Y 8 ID XRODRMPGMNTB. strength=1287 XPB_HUMAN|P19447 ( 74) RPLWVAPDGHIFLEAFSPVYK 54 XPB_MOUSE|P49135 ( 74) RPLWVAPDGHIFLEAFSPVYK 54 P91579 ( 80) RPLYLAPDGHIFLESFSPVYK 67 XPB_DROME|Q02870 ( 84) RPLWVAPNGHVFLESFSPVYK 79 RA25_YEAST|Q00578 ( 131) PLWISPSDGRIILESFSPLAE 100 Q38861 ( 52) RPLWACADGRIFLETFSPLYK 71 O13768 ( 90) PLWINPIDGRIILEAFSPLAE 100 O00835 ( 79) RPIWVCPDGHIFLETFSAIYK 86 library(Biostrings).5%=985.c("RPLWVAPDGHIFLEAFSPVYK") y <. BLOCK AC PR00851A. Conserved region.data(BLOSUM50) x <. substitutionMatrix = "BLOSUM5 > pairwiseAlignment(AAString(x).c("PLWISPSDGRIILESFSPLAE") x == y pairwiseAlignment(AAString(x). width=21. distance from previous block=(52. 99.131) DE Xeroderma pigmentosum group B protein signature BL adapted. getTrans) > table(ccnd3prot[[1]]) * A C D E F G H I K L 1 31 12 12 21 6 14 7 10 10 41 > table(ccnd3prot[[2]]) M 9 N P Q R S T V 1 17 16 22 19 18 15 W 3 Y 8 * A C D E F G H I K L M N P Q R S T V 1 30 12 12 21 6 14 7 10 10 41 9 1 17 16 22 20 18 15 # Hence. seqs=8.252 APPENDIX A. substitutionMatrix = "BLOSU Global Pairwise Alignment .

org/biocLite. Plot of CG proportion from Celegans.character(Celegans$chrI[i:(i+100)]) plot(GCperc.R") biocLite("BSgenome.UCSC.character(Celegans$chrI))) * (1/4)^6 [1] 3681. Celegans$chrI.UCSC.Celegans. How many exact matches has Chromosome I of Celegans.ce2) along a window of 100 nucleotides.759 9.253 1: RPLWVAPDGHIFLEAFSPVYK 2: RPLWVAPDGHIFLEAFSPVYK Score: 154 > > z <.gap Global Pairwise Alignment 1: RPLWVAP-DGHIFLEAFSPVYK 2: -PLWISPSDGRIILESFSPLAE Score: 85 8. AAString(z). library(seqinr) source("http://bioconductor.Celegans. max.c("PLWISPSDGRIILESFSPLAE") > > x == y [1] TRUE > pairwiseAlignment(AAString(x)."gatatc" > countPattern(subseq.ce2") library(BSgenome.double() for (i in 1:10000) GCperc[i] <.000 nucleotides.type="l") (b) A binding sequence of the enzyme EcoRV is the subsequence GATATC. (a) Produce a plot of the CG proportion of the chromosome I of Celegans (Celegans. .ce2) GCperc <.GC(s2c(as.mismatch = 0) [1] 3276 > length(s2c(as.UCSC. > subseq <. Plot of codon usage. substitutionMatrix = "BLOSUM50". Take the ﬁrst 10. 6875 0.] 0. as.as. Visualize by a transition graph the following transition matrices.vector)) row. 2. The answers are provided by the following.2.uco.2] [1.sapply(ccndhs$req. Computing probabilities.lapply(ccnd3.as.data. getSequence) ccnd.names(df) <.frame(lapply(ccnd.uco <.375 > P %*% P [.uco[[1]]) global <.vector)) row.names(df) <.1/4.byrow=T) > pi0 <.] 0.rowSums(df) title <.25 [2. uco.lapply(ec999. 1.2] [1. coli coding sequences" dotchart. uco.] 0.1/2).3125 [2.50 .254 APPENDIX A."Codon usage in 999 E.75 0.2. index="eff") df <. ANSWERS TO EXERCISES data(ec999) ec999.frame(lapply(ec999.1] [. as.uco(global.] 0. main = title) choosebank("genbank").50 0.rowSums(df) title <.names(ccnd.1/2.uco."Codon usage in ccnd3 homo sapiens coding sequences" dotchart.1/2) > pi0 %*% P [.3750 > P [.625 0. > P <.] 0. library(seqinr) query("ccndhs". main = title) Answers to exercises of Chapter 10: Markov Models.uco(global.data.c(1/2.uco[[1]]) global <.1] [.uco <.names(ec999."sp=homo sapiens AND k=ccnd@") ccnd <.6250 0.matrix(c(3/4.2] [1. index="eff") df <. Consult your teacher.1] [.

3 epsilon <.gamma * piA.1] <. (a) Program the rate matrix in such a manner that it is simple to adapt for other values of the parameters.0.02311107 [2.4] [1.4. Q[3.15. zeta <.0.400 0.64908639 0.4.045 [4. piT <.matrix(data=NA.] 0.070 1.105 0.530 (b) The transversion rate is larger then the transition rate because the blocks outside the main diagonal have lower values.alpha * piG.2] [.0. πC = 0.3] <.15.] 0.alpha * piA. = 0.51569256 0. Q[2.0 diag(Q) <. gamma <.0.2] <.beta * piA. gamma * piT delta * piC.35.0.3] Q[1.060 0. Q[4.-apply(Q. delta <.1115233 0.4] <<<<beta * piC. Q[4.delta * piG.4] [1.zeta * piC diag(Q) <.3.4 Q <. (c) The probability transition matrix is > P <.1] <.] 0.1] [.5.1.0. epsilon * piT Q[3.] 0.2. γ = 0. piC <.0.3] Q[2.4] <.400 -1.1] <.01841667 .] 0.225 0.735 0.35.] -1.1392058 0.4] Q[2. β = 0.Matrix(Q) > Q 4 x 4 Matrix of class "dgeMatrix" [.as.4.2] <.35.175 0.15 alpha <. Q[1. Programming GTR. Use πA = 0. Q[3.22097363 0. πT = 0.255 3. beta <. piG <. library(Matrix) piA <.635 1.060 [2.4.600 -0.matrix(expm(Q)) > P [. α = 4.075 0.2] <.4) Q[1. and ζ = 4.0.105 -0.2. πG = 0.epsilon* piG.15.sum) Q <.3] [.2] [.030 [3.32199057 0. δ = 0.35.3] [.delta* pi Q[4.1] [.5.

model = "raw") p <.sep="") seqbin <.character = TRUE in the read.P.04621015 0."g". species.StateSpace <.256 APPENDIX A.read.replace=T.character = FA Down load the sequences AJ534526 and AJ534527."c".1.99) 4.matrix(0.markov2(StateSpace.names = TRUE.pi0) for(k in 1:(n-1)){ seq[k+1] <.nc=1) seq[1] <.GenBank function.sample(StateSpace.6397090 0.1/4.02244359 [4."t") pi0 <.replace=T. ANSWERS TO EXERCISES [3.1.05203969 0.GenBank(accnr. (b) Two solution of computing the proportion of diﬀerent nucleotides are dist.sample(StateSpace. (a) accnr <.c(1/4. Distance according to JC69.P.n){ seq <.1/4) markov2 <.08457814 0. Hint: Use as.1/4.8263804 0. as.09913633 0.22950271 rownames(P) <.function(StateSpace.nr=n.] 0.-log(1-4*p/3)*3/4.sum(seq$AJ534526 != seq$AJ534527)/1143 (c) Simply insert the obtained p in the formula d <.paste("AJ5345".26:27. .] 0.dna(seqbin.c("a".P[seq[k return(seq) } seq <.colnames(P) <.

(1988).M. Lobry. Beran. A.. Evidence that microRNA precursors. J. R. Chambers.S. B. J. Asymptotic theory for bootstrap methods in statistics. Wuyts. A Simple Test for Heteroscedasticity and Random Coeﬃcient Variation. A.J. Vignetti. J. and Van de Peer. T. J. G. 29112917. e Breiman. Gentleman. R. New Jersey: Bell Telephone Laboratories. Introduction to probability and mathematical statistics. & Ducharme. Mandelli.. (2008). and C. The Annals of Statistics. L. eds. Bonnet. J. Palmeira. A. M. (1979). (1991). R. S.r-project. Introductory statistics with R. 13. T. J..R. L. Breusch.Appendix B References Dalgaard. & Rouze. Bootstrap tests and conﬁdence regions for functions of a covariance matrix.org/. Monterey: Wadsworth. Penel. & Srivastava.S. Paciﬁc Grove: Duxbury. Chambers. A. J. & Pagan A. L. P. & Hastie. H. New York: Springer.. Stone. & Wilks. Olshen... L. Vitale. (1992). M.r-forge. Necxsulea. (1984) Classiﬁcation and Regression Trees. Xiaochun Li. R. 95115. M. P. and Foa R. SeqinR 2. (1985).J. URL: http://seqinr. Chiaretti. Humblot. Paciﬁc Grove: Wadsworth and Brooks/Cole.A. Beran. Econometrica 47. Y.M. (1992) Statistical Models in S. D. (2004) Gene expression proﬁle of adult T-cell 257 .. Becker. have lower folding free energies than random sequences Bioinformatics..0-1: a contributed package to the project for statistical computing devoted to biological sequences retrieval and analysis. E.R. Charif. The new S language. unlike other non-coding RNAs. Ritz. F.R. Bain. (2004).R. 20. (2002). S. & Engelhardt. Montreal: Centre de recherche math´matique.. Friedman. 12871294.

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L. and Stephens. A. (1995) A Remark on Algorithm AS 181: The W Test for Normality. New Jersey: Pearson Education. (1981). Austria.C. Palmeira. S. K. B. & Toutenburg (1995). T. Paciﬁc Grove.B. Royston. Molecular Biology and Evolution. E. routines. R Development Core Team (2009). Bioinformatics and functional genomics. & Witmer. Paradis. (2006) UV-targeted dinucleotides are not depleted in light-exposed Prokaryotic genomes.. New York: Springer. Applied Statistics. 35. In: D’Agostino. R: A language and environment for statistical computing. Analysis of Phylogenetics and Evolution with R.. & Miller. Ripley. L. 547551. R. J. M.A. Posfai. Numerical recipes in Pascal. Paciﬁc Grove: Duxbury.. New Jersey: Prentice Hall.P. Stephens. (1980).. Linear Models. B. Marazzi. (1986): Tests based on EDF statistics. E. R.R. M. D. J. 44. The melanoma antigen gene (MAGE) family is clustered in the chromosomal band Xq28. and S functions for robust statistics. (1999).. 9.260 APPENDIX B. URL http://www. 23:2214-2219. B. and Lobry.J. Pollard. Vienna. B. 10. Marcel Dekker. 337349... & Vettering W. (1995). Samuels. W. Teukolsky. Exact type 1 error rates for robustness of Student’s t=testwith unequal variances.org. Steck. Press. REBASE–enzymes and genes for DNA restriction and modiﬁcation.A.H. U. Guguen.. Annals of statistics.T. Poustka. (1992). Algorithms. R Foundation for Statistical Computing. J. 5. New York. Cambridge: Cambridge University Press. Freund’s Mathematical Statistics. Flannery. Pattern Recognition and Neural Networks. CA.D. (2000) Fundamentals of Biostatistics.L. M. Rogner.. (2003) Statistics for the Life Sciences.R.: Goodness-of-Fit Techniques. eds. Macelis.A. New York: Springer. Ramsey.29(3):725-31.. D. Wadsworth & Brooks/Cole. (1996)..H. C. (2003). REFERENCES Miller. Roberts. Strong consistency of K-means clustering. Genomics. Nucleic Acids Res. (1993). J. Rosner. Vincze. Pevsner. M. (2007). Wilke.R-project. Journal of educational statistics. I. Rao. P. 135-140. . A.A. John E. P. ISBN 3-900051-07-0. New York: Wiley-Liss. New York: Cambridge University press. (2006). Korn.

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262 APPENDIX B. REFERENCES .

35 263 . 101 Needleman-Wunsch. 57 Fisher test. 74 matrix computations. 130 data matrix. 62 frequency table. 2 installing R.Index aggregation. 25 k-means cluster analysis. 107 gol. 104 background correction. 158 mismatch. 24 median absolute deviation. 12 help. 185 bootstrap. 64 annotation. 174 F-distribution. 6 data vector. 4 correlation coeﬃcient. 150 confusion table. 1 installing Bioconductor. 41 design matrix. 176 distance. 184 neural network. 11 Golub et al. 40 F-test. 17 genBank. 3 histogram. 94 Binomial test. 162 normal distribution. 158 construct a sequence. 18 gene ﬁltering. 85 install R. (1999) data. 127. 125 Kruskal-Wallis test. 10 grep. 59 classiﬁcation tree. 4 chi-squared distribution. 107 GO. 58 BLOSUM50. 5 density. 91 model matrix. 87 linear model. 97 gene ontology. 95 Anderson-Darling test. 2 interquartile range. 118 downloading sequences. 8 mean. 25 misclassiﬁcation rate. 20 calculator.fac. 19 homoscedasticity. 132 box-and-wiskers-plot. 37 chi-squared test. 24 median. 101 dinucleotide.

19 support vector machine. 159 Wilcoxon rank test. 94 one sample t-test. 13 sample variance. 147 standard deviation. 2 perfect match. 65 Z-test. 63 normalization. 51 one sided hypothesis. 25 stripchart. 48 one-way analysis of variance. 148 rma. 22 quartile. 20 query language. 77 packages. 39 training set. 85 normality test. 159 triangle inequality. 54 validation set. 48 INDEX . 25 sensitivity. 147 principal components analysis. 133 Quantile-Quantile plot. 95 running scripts. 48 two-sample t-test. 118 two sided hypothesis. 48 single linkage cluster analysis. 121 speciﬁcity. 161 T-distribution. 173 receiver operator curve.264 normality of residuals. 91 Phylogenetic tree. 63 signiﬁcance level. 147 Shapiro-Wilk test. 203 predictive power.