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Principal Component Analysis with published CLL gene expression data (Herishanu et SARS-CoV-2 spike protein
al, Blood 2011) variant - 22/01/2021
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Over the last few years, my understanding of chronic lymphocytic leukaemia has developed. Leukaemic cell proliferation has been shown to be a
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key element of the disease. The site of CLL cell proliferation was an important question. A gene expression analysis published in Blood by
Tuesday... - 18/05/2020
Herishanu et al in 2011 showed a proliferative gene expression pattern in the leukeamic cells from the lymph nodes as distinct from other sites of
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the body (peripheral blood or bone marrow). Tidy Tuesday... - 30/04/2020

I've been exploring the gene expression data using R. As a first step, I've explored the principal component analysis similar to that shown in Figure
1A of the paper. Follow by Email

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The PCA is convincing because the three groups of samples cluster among themselves. The 3D plot is useful because the three components
separate the groups while two dimensions do not. ► October (1)
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Principal Component Analysis with
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1 sur 7 01/03/2021 à 13:02

R for Biochemists: Principal Component Analysis with publi... https://rforbiochemists.blogspot.com/2016/06/principal-comp...

There are other ways of visualising PCA plots... Sources are indicated as part of the script. Quick-R: Home Page

Here is the script that I used to analyse this:

SCRIPT START
# install.packages("RCurl", "scatterplot3d")

library(RCurl)
library(scatterplot3d)

# A few year's ago a gene expression profile paper was published in Blood by Herishanu et al
# that gave us a new insight into chronic lymphocytic leukamia.
# http://www.bloodjournal.org/content/117/2/563.long
# I have written this script to explore the data
# and to reproduce the principal component analysis.

# I have downloaded the original CEL files from GEO

# http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE21029
# they were placed in a folder and the expression values extracted
# what's produced is Affymetrix IDs and log2 expression values

# I have created two data sets

# we can reproduce the PCA workflow with a subset of the data is required (5000 genes selected randomly).
# this will be faster to download and visualise.
# get the data - needs connection to the internet
# to use remove the hashtag "#"
# x <- getURL("https://raw.githubusercontent.com/brennanpincardiff/RforBiochemists/master
/data/herishanuMicroArrayDataSubset.tsv")

# here is a link for all the data (>50000 probes)

x <- getURL("https://raw.githubusercontent.com/brennanpincardiff/RforBiochemists/master
/data/herishanuMicroArrayData.tsv")

# turn the data into a useful data.frame

data <- read.table(text = x, header = TRUE, sep = "\t")

# read in a file with names of samples

# created this from cut and paste of web page combined with manipulation in Word & Excel
sampID <- read.csv("https://raw.githubusercontent.com/brennanpincardiff/RforBiochemists/master
/data/Herishanu_Samp_ID.csv", header = FALSE)

str(sampID)
sampID$V1 <- as.character(sampID$V1)

# is order of column names same as list in sampID?

data.col.name <- colnames(data)
data.col.name <- gsub(".CEL", "", data.col.name)
sampID.name <- sampID$V1
all.equal(data.col.name, sampID.name)
# TRUE so that is useful.

# change column names from chip to PB, BM and LN

# for peripheral blood, bone marrow and lymph node respectively
sampID$Name <- paste0(as.character(sampID$V3), as.character(sampID$V5))
colnames(data) <- sampID$Name

# how do the sample cluster?

# I first need to find the "distances" between the arrays
# and show those distances in the hierarchical cluster plot
# I can pile up all the commands in one
# for more of a step by step approach, see these scripts:
# http://rforbiochemists.blogspot.co.uk/2016/02/visualising-some-cll-proteomic-data-for.html
# http://rforbiochemists.blogspot.co.uk/2016/03/gene-expression-analysis-and.html

plot(hclust(dist(t(data))))

# this is interesting because all the patient samples cluter together!

# with bone marrow and peripheral blood closer together than LN in all cases.
# this is what it says in the paper and is shown in the supplementary data.

2 sur 7 01/03/2021 à 13:02

R for Biochemists: Principal Component Analysis with publi... https://rforbiochemists.blogspot.com/2016/06/principal-comp...

# http://www.bloodjournal.org/content/117/2/563/tab-figures-only

# What's needed is a way to normalise by patient - HOW??

# Quote from the paper:
# "In the 12 patients in whom all 3 compartments had been arrayed,
# the patient effect on gene expression was subtracted
# by mean centering the expression value of each gene across the 3 compartments
# for each patient separately."

# the twelve with all 3 compartments: #1, #2, #3, #4, #8, #9, #10, #11, #12, #13, #25, #26

# so if I understand this correctly, I need to extract a patient sample

# then mean centre all three samples,
# and repeat for all 12 samples

# let's explore this concept with just two samples to see if it works
# pick out data for patient #26
samp26 <- data[,grep("#26", sampID$Name)]

# pick out data for patient #25

samp25 <- data[,grep("#25", sampID$Name)]

# put them together

twosamp <- cbind(samp26, samp25)

# visualise with a box plot - look normalised

boxplot(twosamp)

plot(hclust(dist(t(twosamp))))

# cluster by patient

# I think I understand what they have done.

# mean centred for each gene.... i.e. by row in the data
# For us this is really each probeset
# http://gastonsanchez.com/how-to/2014/01/15/Center-data-in-R/

# for each of the values substract the rowMeans

samp25.s <- samp25 - rowMeans(samp25)
samp26.s <- samp26 - rowMeans(samp26)
twosamp.s <- cbind(samp26.s, samp25.s)

plot(hclust(dist(t(twosamp.s))))

# This seems to work and gives a different cluster diagram

# with LN coming together nicely.

3 sur 7 01/03/2021 à 13:02

R for Biochemists: Principal Component Analysis with publi... https://rforbiochemists.blogspot.com/2016/06/principal-comp...

# apply this to the whole data set.

# extract each patient cohort
# these twelve were: #1, #2, #3, #4, #8, #9, #10, #11, #12, #13, #25, #26
# do #1 and # 2 first then do the other automatically...

# pat # 1
colnames(data)
pat_1 <- cbind(data[,1], data[,27], data[,46])
colnames(pat_1) <- c("PB#1", "BM#1", "LN#1")
pat_1.s <- pat_1 - rowMeans(pat_1)
# pat # 2
pat_2 <- cbind(data[,2], data[,28], data[,47])
colnames(pat_2) <- c("PB#2", "BM#2", "LN#2")
pat_2.s <- pat_2 - rowMeans(pat_2)

data.n <- cbind(pat_1.s, pat_2.s)

data.reqd <- c("#3", "#4", "#8", "#9", "#10", "#11", "#12", "#13", "#25", "#26")
for(i in 1:length(data.reqd)){
samp <- data[,grep(data.reqd[i], sampID$Name)]
samp <- samp - rowMeans(samp)
data.n <- cbind(data.n, samp)
}

# now have a file called dat.exp.n - normalised within patients.

colnames(data.n)
plot(hclust(dist(t(data.n))))

# nice cluster by region with good separation of LN samples

# some overlap within PB and LN

# extract cell site to use for colours in PCA plots

names <- colnames(data.n)
colourby <- gsub("#\\d+", "", colnames(data.n))
colourby <- gsub("\\.", "", colourby)
colourby <- gsub("\\d+", "", colourby)

# this does cluster the LN samples distinctly but there is still some overlap
# of Peripheral Blood and Bone Marrow.
# PCA from the paper looks nice and convincing
# http://www.r-bloggers.com/computing-and-visualizing-pca-in-r/
# uses function princomp()

exp.pca <- princomp(data.n) # this function does the PCA

print(exp.pca)
plot(exp.pca, type = "l")

Plot of variance by component for CLL gene expression patterns

plot(princomp(data.n)$loadings)

4 sur 7 01/03/2021 à 13:02

R for Biochemists: Principal Component Analysis with publi... https://rforbiochemists.blogspot.com/2016/06/principal-comp...

A simple 2D plot of component 1 vs component 2

# http://www.r-bloggers.com/visualizing-principal-components/
p <- princomp(data.n)
loadings <- p$loadings[]
p.variance.explained <- p$sdev^2 / sum(p$sdev^2)

# plot percentage of variance explained for each principal component

barplot(100*p.variance.explained, las=2, xlab='', ylab='% Variance Explained')

Looking at the effects of each component as a bar plot.

#*****************************************************************
# 2-D Plot
#******************************************************************
x <- loadings[,1]
y <- loadings[,2]
z <- loadings[,3]
cols <- as.factor(colourby)
cols <- gsub("PB", "green", cols)
cols <- gsub("BM", "red", cols)
cols <- gsub("LN", "blue", cols)

# pch = 24: triangle point-up

# pch = 22: square
# pch = 21: circle
symbols <- as.factor(colourby)
symbols <- gsub("PB", 24, symbols)
symbols <- gsub("BM", 21, symbols)
symbols <- gsub("LN", 22, symbols)
symbols <- as.numeric(symbols)

# plot loadings on the first and second principal components

# identify sample by body location
plot(x, y, type='p',
pch=symbols,
xlab='Comp.1', ylab='Comp.2',
col = cols, main = "Principal Component Analysis - CLL cell gene expression")

A simple 2D plot of component 1 vs component 2

Each sample is coloured by location

# add a legend to the top of the plot

legend("top", # location
bty="n", # suppress legend box, shrink text 50%
title="Body Location of sample",

5 sur 7 01/03/2021 à 13:02

R for Biochemists: Principal Component Analysis with publi... https://rforbiochemists.blogspot.com/2016/06/principal-comp...

c("PB", "BM", "LN"),

pch=symbols, col = cols, horiz=TRUE)

# label up the points

text(x, y, colnames(data.n), col=cols, cex=.5, pos=4)

# Comment: lymph node samples separate nicely from other samples

# some overlap between bone marrow and peripheral blood

#*****************************************************************
# 3-D Plot, for good examples of 3D plots
# http://statmethods.wordpress.com/2012/01/30/getting-fancy-with-3-d-scatterplots/
#******************************************************************
# plot all companies loadings on the first, second, and third principal components and highlight points
according to the sector they belong
s3d = scatterplot3d(x, y, z,
xlab='Comp.1', ylab='Comp.2', zlab='Comp.3',
color=cols, pch = symbols,
main = "Principal Component Analysis in 3D \n CLL cell gene expression")

A 3D graph separates the different groups of samples.

s3d.coords = s3d$xyz.convert(x, y, z)
text(s3d.coords$x, s3d.coords$y,
labels=colnames(data.n), col=cols, cex=.8, pos=4)

Labels can be added but I'm not sure they help in this example.

# change the order and the angle to make it look a bit more like the figure
s3d = scatterplot3d(y, x, z,
xlab='PC2', ylab='PC1', zlab='PC3',
color=cols, pch = symbols,
angle=-25,
grid = FALSE,

6 sur 7 01/03/2021 à 13:02

R for Biochemists: Principal Component Analysis with publi... https://rforbiochemists.blogspot.com/2016/06/principal-comp...

main = "Principal Component Analysis in 3D \n CLL cell gene expression")

par(xpd=TRUE) # allows legend outside the graph

# add legend
legend(10, -4.5, # location
bty="n", # suppress legend box
title="Site of sample",
c("PB", "BM", "LN"),
pch=symbols, col = cols, horiz=TRUE)

# add source as text

text(4, -6.5, cex =0.7,
"Source: Herishanu et al, Blood 2011 117:563-574; doi:10.1182/blood-2010-05-284984")

SCRIPT END

Posted by Paul Brennan at 13:23

Labels: 3D, cll, cluster, clustering, Herishanu, PCA, published data

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