MicroarrayDataAnalysis

StuartM.Brown
NYUSchoolofMedicine

TheCentralDogmaofMolecularBiology
DNAistranscribedintoRNAwhichisthen
translatedintoprotein

DNA

transcription

RNA

translation

protein

replication

Measured by Microarray

WhatisaMicroarray
Asimpleconcept:DotBlot+Northern
Reversethehybridizationputtheprobes
onthefilterandlabelthebulkRNA
Makeprobesforlotsofgenesamassively
parallelexperiment
Makeittinysoyoudontneedsomuch
RNAfromyourexperimentalcells.
Makequantitativemeasurements

Microarrays are Popular

At NYU Med Center we are now collecting

about 3 GB of microarray data per week (60

chips, 6-10 different experiments)
PubMed search "microarray"= 13,948 papers
2005
2004
2003
2002
2001
2000

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4406
3509
2421
1557
834
294

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4500

4406

4000
3509

3500
3000
2500

2421

2000
1557

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1000
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834
294

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2001

2002

2003

2004

2005

AFilterArray

DNAChipMicroarrays
Putalargenumber(~100K)ofcDNAsequencesor
syntheticDNAoligomersontoaglassslide(orother
subtrate)inknownlocationsonagrid.
LabelanRNAsampleandhybridize
MeasureamountsofRNAboundtoeachsquareinthe
grid
Makecomparisons
Cancerousvs.normaltissue
Treatedvs.untreated
Timecourse

Manyapplicationsinbothbasicandclinicalresearch

cDNAMicroarrayTechnologies
SpotclonedcDNAsontoaglassmicroscope
slide
usuallyPCRamplifiedsegmentsofplasmids

Label2RNAsampleswith2differentcolorsof
flourescentdyecontrolvs.experimental
MixtwolabeledRNAsandhybridizetothe
chip
Maketwoscansoneforeachcolor
Combinetheimagestocalculateratiosof
amountsofeachRNAthatbindtoeachspot

Spot your own Chip

(plans available for free from Pat Browns website)

Robot spotter

Ordinary glass
microscope slide

CombinescansforRed&Green

False color image is made from digitized fluorescence data,

not by superimposing scanned images

cDNASpottedMicroarrays

AffymetrixGenechipsystem
Uses25baseoligossynthesizedinplaceona
chip(20pairsofoligosforeachgene)
RNAlabeledandscannedinasinglecolor
onesampleperchip

Canhaveasmanyas20,000genesonachip
Arraysgetsmallereveryyear(moregenes)
Chipsareexpensive
Proprietarysystem:blackboxsoftware,can
onlyusetheirchips

Affymetrix Gene Chip

AffymetrixTechnology

Affymetrix Pivot Table

normal
tumor
tumor
normal
normal
tumor
ID_REF
VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL
AFFX-BioB-5_at
210.6 P
234.6 P
362.5 P
389 P
305.6 P
330.5 P
AFFX-BioB-M_at
393 P
327.8 P
501.4 P
816.5 P
542 P
440.8 P
AFFX-BioB-3_at
264.9 P
164.6 P
244.7 P
379.7 P
261.3 P
303.7 P
AFFX-BioC-5_at
738.6 P
676.1 P
737.6 P
1191.2 P
917 P
767.9 P
AFFX-BioC-3_at
356.3 P
365.9 P
423.4 P
711.6 P
560.3 P
484.9 P
AFFX-BioDn-5_at
566.3 P
442.2 P
649.7 P
834.3 P
599.1 P
606.9 P
AFFX-BioDn-3_at
3911.8 P
3703.7 P
4680.9 P
6037.7 P
4653.7 P
4232 P
AFFX-CreX-5_at
6433.3 P
5980 P
7734.7 P
10591 P
8162.1 P
8428 P
AFFX-CreX-3_at
11917.8 P
9376.7 P
11509.3 P
16814.4 P
13861.8 P
13653.4 P
AFFX-DapX-5_at
12.2 A
44.3 M
31.2 A
37.7 P
33.3 A
12.8 A
AFFX-DapX-M_at
57.8 M
42.5 A
79 M
48.8 P
39.5 A
39.2 A
AFFX-DapX-3_at
29.8 A
6.2 A
23.4 A
28.4 A
3.2 A
7.6 A
AFFX-LysX-5_at
15.3 A
16.2 A
15.6 A
16.7 A
3.1 A
3.9 A
AFFX-LysX-M_at
33.2 A
12 A
17.7 A
37.3 A
49.2 A
9.1 A
AFFX-LysX-3_at
40.7 M
10.7 A
36.2 A
22.1 A
22.8 A
28.2 A
AFFX-PheX-5_at
7.8 A
3 A
7.6 A
5.6 A
5 A
6.4 A
AFFX-PheX-M_at
4.2 A
4.8 A
6.8 A
6.1 A
3.7 A
5.5 A
AFFX-PheX-3_at
54.2 A
39.6 A
19.4 A
16.1 A
44.7 A
31.2 A
AFFX-ThrX-5_at
8.2 A
11.2 A
13.2 A
9.5 A
8.5 A
7.5 A
AFFX-ThrX-M_at
38.1 A
30.6 A
37.6 A
7.2 A
26.9 A
36.3 A
AFFX-ThrX-3_at
15.2 A
5 A
15 A
8.3 A
36.8 A
11.5 A
AFFX-TrpnX-5_at
11.2 A
11.8 A
22.2 A
22.1 A
8.9 A
35.6 A
AFFX-TrpnX-M_at
9 A
8.1 A
9.1 A
8.7 A
8.1 A
12 A
AFFX-TrpnX-3_at
19.8 A
12.8 A
11.8 A
43.2 M
17.4 A
10 A
AFFX-HUMISGF3A/M97935_5_at
82.7 P
120.7 P
92.7 P
46.4 P
55.9 P
46.5 P
AFFX-HUMISGF3A/M97935_MA_at
397.6 P
416.7 P
244.8 A
181.4 A
197.5 A
192.3 A
AFFX-HUMISGF3A/M97935_MB_at
206.2 P
303 P
300.8 P
253.5 P
195.3 P
216 P
AFFX-HUMISGF3A/M97935_3_at
663.8 P
723.9 P
812.1 P
666.1 P
629.4 P
754.1 P
AFFX-HUMRGE/M10098_5_at
547.6 P
405.9 P
6894.7 P
3496.1 P
1958.5 P
5799.4 P
AFFX-HUMRGE/M10098_M_at
239.1 P
175.8 P
3675 P
1348.6 P
695.9 P
2428.2 P
AFFX-HUMRGE/M10098_3_at
1236.4 P
721.4 P
9076.1 P
7795.9 P
4237.1 P
7890 P
AFFX-HUMGAPDH/M33197_5_at
19508 P
19267.1 P
22892 P
26584 P
29666.6 P
25038.1 P
AFFX-HUMGAPDH/M33197_M_at
18996.6 P
20610.4 P
21573.7 P
29936 P
30106.6 P
22380.2 P
AFFX-HUMGAPDH/M33197_3_at
18016.4 P
17463.8 P
20921.3 P
26908.3 P
28382.2 P
21885 P
AFFX-HSAC07/X00351_5_at
23294.6 P
21783.7 P
18423.3 P
21858.9 P
23517.1 P
19450.3 P
AFFX-HSAC07/X00351_M_at
25373.1 P
24922.8 P
22384.2 P
25760.2 P
27718.5 P
21401.6 P
AFFX-HSAC07/X00351_3_at
20032.8 P
20251.1 P
20961.7 P
23494.6 P
23381.2 P
21173.3 P

DataAcquisition

Scanthearrays
Quantitateeachspot
Subtractbackground
Normalize
Exportatableoffluorescentintensities
foreachgeneinthearray

Automate!!
Allofthiscanbedoneautomaticallyby
software.
Muchmoreconsistent
Mistakeswillbemade(especiallyinthe
spotquantitation)butyoucant
manuallycheckhundredsofthousands
ofspots

AffymetrixSoftware
AffymetrixSystemistotallyautomated
Computesasinglevalueforeachgenefrom40
probes(usingsurprisinglykludgymath)
Highlyreproducible
(rescanofsamechiporhyb.ofduplicatechipswith
samelabeledsamplegivesverysimilarresults)

Incorporatesfalseresultsduetoimageartefacts
dust,bubbles
pixelspilloverfrombrightspottoneighboringdark
spots

Goals of a Microarray
Experiment
1. Find the genes that change expression

between experimental and control

samples
2. Classify samples based on a gene
expression profile
3. Find patterns: Groups of biologically
related genes that change expression
together across samples/treatments

BasicDataAnalysis
Foldchange(relativeincreaseordecreasein
intensityforeachgene)
Setcutofffilterforlowvalues
(background+noise)
Clustergenesbysimilarchangesonlyreally
meaningfulacrossmultipletreatmentsor
timepoints
Clustersamplesbysimilargeneexpression
profiles

Streamlined Affy Analysis

normal
tumor
tumor
normal
normal
tumor
ID_REF
VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL VALUE
ABS_CALL
AFFX-BioB-5_at
210.6 P
234.6 P
362.5 P
389 P
305.6 P
330.5 P
AFFX-BioB-M_at
393 P
327.8 P
501.4 P
816.5 P
542 P
440.8 P
AFFX-BioB-3_at
264.9 P
164.6 P
244.7 P
379.7 P
261.3 P
303.7 P
AFFX-BioC-5_at
738.6 P
676.1 P
737.6 P
1191.2 P
917 P
767.9 P
AFFX-BioC-3_at
356.3 P
365.9 P
423.4 P
711.6 P
560.3 P
484.9 P
AFFX-BioDn-5_at
566.3 P
442.2 P
649.7 P
834.3 P
599.1 P
606.9 P
AFFX-BioDn-3_at
3911.8 P
3703.7 P
4680.9 P
6037.7 P
4653.7 P
4232 P
AFFX-CreX-5_at
6433.3 P
5980 P
7734.7 P
10591 P
8162.1 P
8428 P
AFFX-CreX-3_at
11917.8 P
9376.7 P
11509.3 P
16814.4 P
13861.8 P
13653.4 P
AFFX-DapX-5_at
12.2 A
44.3 M
31.2 A
37.7 P
33.3 A
12.8 A
AFFX-DapX-M_at
57.8 M
42.5 A
79 M
48.8 P
39.5 A
39.2 A
AFFX-DapX-3_at
29.8 A
6.2 A
23.4 A
28.4 A
3.2 A
7.6 A
AFFX-LysX-5_at
15.3 A
16.2 A
15.6 A
16.7 A
3.1 A
3.9 A
AFFX-LysX-M_at
33.2 A
12 A
17.7 A
37.3 A
49.2 A
9.1 A
AFFX-LysX-3_at
40.7 M
10.7 A
36.2 A
22.1 A
22.8 A
28.2 A
AFFX-PheX-5_at
7.8 A
3 A
7.6 A
5.6 A
5 A
6.4 A
AFFX-PheX-M_at
4.2 A
4.8 A
6.8 A
6.1 A
3.7 A
5.5 A
AFFX-PheX-3_at
54.2 A
39.6 A
19.4 A
16.1 A
44.7 A
31.2 A
AFFX-ThrX-5_at
8.2 A
11.2 A
13.2 A
9.5 A
8.5 A
7.5 A
AFFX-ThrX-M_at
38.1 A
30.6 A
37.6 A
7.2 A
26.9 A
36.3 A
AFFX-ThrX-3_at
15.2 A
5 A
15 A
8.3 A
36.8 A
11.5 A
AFFX-TrpnX-5_at
11.2 A
11.8 A
22.2 A
22.1 A
8.9 A
35.6 A
AFFX-TrpnX-M_at
9 A
8.1 A
9.1 A
8.7 A
8.1 A
12 A
AFFX-TrpnX-3_at
19.8 A
12.8 A
11.8 A
43.2 M
17.4 A
10 A
AFFX-HUMISGF3A/M97935_5_at
82.7 P
120.7 P
92.7 P
46.4 P
55.9 P
46.5 P
AFFX-HUMISGF 3A/M97935_MA_at
397.6 P
416.7 P
244.8 A
181.4 A
197.5 A
192.3 A
AFFX-HUMISGF 3A/M97935_MB_at
206.2 P
303 P
300.8 P
253.5 P
195.3 P
216 P
AFFX-HUMISGF3A/M97935_3_at
663.8 P
723.9 P
812.1 P
666.1 P
629.4 P
754.1 P
AFFX-HUMRGE/M10098_5_at
547.6 P
405.9 P
6894.7 P
3496.1 P
1958.5 P
5799.4 P
AFFX-HUMRGE/M10098_M_at
239.1 P
175.8 P
3675 P
1348.6 P
695.9 P
2428.2 P
AFFX-HUMRGE/M10098_3_at
1236.4 P
721.4 P
9076.1 P
7795.9 P
4237.1 P
7890 P
AFFX-HUMGAPDH/M33197_5_at
19508 P
19267.1 P
22892 P
26584 P
29666.6 P
25038.1 P
AFFX-HUMGAPDH/M33197_M_at
18996.6 P
20610.4 P
21573.7 P
29936 P
30106.6 P
22380.2 P
AFFX-HUMGAPDH/M33197_3_at
18016.4 P
17463.8 P
20921.3 P
26908.3 P
28382.2 P
21885 P
AFFX-HSAC07/X00351_5_at
23294.6 P
21783.7 P
18423.3 P
21858.9 P
23517.1 P
19450.3 P
AFFX-HSAC07/X00351_M_at
25373.1 P
24922.8 P
22384.2 P
25760.2 P
27718.5 P
21401.6 P
AFFX-HSAC07/X00351_3_at
20032.8 P
20251.1 P
20961.7 P
23494.6 P
23381.2 P
21173.3 P

Raw data

Significance
t-test
SAM
Rank Product

Normalize

Filter

(RMA)

Classification
PAM
Machine learning
Gene lists

Function

(Genome Ontology)

Present/Absent
Minimum value
Fold change

Clustering

SourcesofVariability
Imageanalysis(identifyingandquantitating
eachspotonthearray)
Scanning(laseranddetector,chemistryofthe
flourescentlabel))

Hybridization(temperature,time,mixing,etc.)
Probelabeling
RNAextraction
Biologicalvariability

Scatterplotofallgenesina
simplecomparisonoftwo
control(A)andtwo
treatments(B:highvs.low
glucose)showingchangesin
expressiongreaterthan2.2
and3fold.

Thomas Hudson, Montreal Genome Center

Normalization
Cancontrolformanyoftheexperimental
sourcesofvariability(systematic,notrandom
orgenespecific)
Bringeachimagetothesameaverage
brightness
Canusesimplemathorfancy
dividebythemean(wholechiporbysectors)
LOESS(locallyweightedregression)

Nosurebiologicalstandards

RMA
RobustMultichipAverage

Bolstad, B.M., Irizarry R. A., Astrand, M., and Speed,

T.P. (2003), A Comparison of Normalization Methods
for High Density Oligonucleotide Array Data Based
on Bias and Variance. Bioinformatics 19(2):185-193

AretheTreatmentsDifferent?
Analysisofmicroarraydatahastendedtofocuson
makinglistsofgenesthatareupordownregulated
betweentreatments
Beforemakingtheselists,askthequestion:
"Arethetreatmentsdifferent?"
Usestandardstatisticalmethodstoevaluateexpression
profilesforeachtreatment(ttestorftest)
Iftherearedifferences,findthegenesmost
responsible
Iftherearenotsignificantoveralldifferences,then
listsofgeneswithlargefoldchangesmayonlyreflect
randomvariability.

Statistics
Whenyouhavevariabilityinmeasurements,
youneedreplicationandstatisticstofindreal
differences
Itsnotjustthegeneswith2foldincrease,
butthosewithasignificantpvalueacross
replicates
Nonparametric(i.e.rank)orpairedvalue
statisticsmaybemoreappropriate

MultipleComparisons
Inamicroarrayexperiment,eachgene(each
probeorprobeset)isreallyaseparate
experiment
Yetifyoutreateachgeneasanindependent
comparison,youwillalwaysfindsomewith
significantdifferences
(thetailsofanormaldistribution)

FalseDiscovery
Statisticianscallfalsepositivesa"type1error"ora
"FalseDiscovery"
FalseDiscoveyRate(FDR)isequaltothepvalueof
thettestXthenumberofgenesinthearray
Forapvalueof0.01X10,000genes
=100falsedifferentgenes
Youcannoteliminatefalsepositives,butbychoosinga
morestringentpvalue,youcankeepthemmanageable
(tryp=0.001)

TheFDRmustbesmallerthanthenumberofreal
differencesthatyoufindwhichinturndependson
thesizeofthedifferencesandvarabilityofthe
measuredexpressionvalues

SAM
SignificanceAnalysisofMicroarrays
Tusher, Tibshirani and Chu (2001): Significance
analysisofmicroarraysappliedtotheionizingradiation
response.PNAS 2001 98: 5116-5121, (Apr 24).
Excel plugin
Free
Permutation based
Most published method of
microarray data analysis

HigherLevel
Microarraydataanalysis

Clusteringandpatterndetection
Dataminingandvisualization
Controlsandnormalizationofresults
Statisticalvalidatation
Linkagebetweengeneexpressiondataandgene
sequence/function/metabolicpathwaysdatabases
Discoveryofcommonsequencesincoregulated
genes
Metastudiesusingdatafrommultipleexperiments

TypesofClustering
Herarchical
Linksimilargenes,builduptoatreeofall

SelfOrganizingMaps(SOM)
Splitallgenesintosimilarsubgroups
Findsitsowngroups(machinelearning)

PrincipleComponent
everygeneisadimension(vector),findasingle
dimensionthatbestrepresentsthedifferencesin
thedata

Clusterby
color
difference

GeneSpring

SOM
Clusters

Classification
How to sort samples into two classes

based on gene expression data

Cancer vs. normal
Cancer sub-types
(benign vs. malignant)
Responds well to drug vs. poor
response

(i.e. tamoxifen for breast cancer)

Support Vector Machines

Fat planes: With an infinitely thin plane the

data can always be separated correctly, but
not necessarily with a fat one.
Again if a large margin separation exists,
chances are good that we found something
relevant.

PAM: Prediction Analysis for Microarrays

Class Prediction and Survival Analysis for Genomic Expression Data Mining
Performs sample classification from gene expression data,
via "nearest shrunken centroid method'' of Tibshirani, Hastie, Narasimhan and Chu (2002):
"Diagnosis of multiple cancer types by shrunken centroids of gene expression"
PNAS 2002 99:6567-6572 (May 14).

BioConductor
All of these normalization, statistical,

and clustering methods are available in

a free software package called
BioConductor.
www.bioconductor.org

User hostile command line interface

Uses scripts in the 'R' statistical language
>data(SpikeIn)
>pms<pm(SpikeIn)
>mms<mm(SpikeIn)
>par(mfrow=c(1,2))
>concentrations<matrix(as.numeric(sampleNames(SpikeIn)),20,
+12,byrow=TRUE)
>matplot(concentrations,pms,log="xy",main="PM",ylim=c(30,
+20000))
>lines(concentrations[1,],apply(pms,2,mean),lwd=3)
>matplot(concentrations,mms,log="xy",main="MM",ylim=c(30,
+20000))
>lines(concentrations[1,],apply(mms,2,mean),lwd=3)

Functional Genomics
Take a list of "interesting" genes and

find their biological relationships

Gene lists may come from

significance/classfication analysis of
microarrays, proteomics, or other highthroughput methods

Requires a reference set of "biological

knowledge"

Genome Ontology
How to organize biological

knowledge?
Biologists work on a variety of

different research organisms: yeast,

fruit fly, mouse, human
the same gene can have very
different functions (antennapedia)
and very different names
(sonic hedgehog)

GO
Biologists got together a few years ago

and developed a sensible system called

Genome Ontology (GO)
3 hierarchical sets of terminology
Biological Process
Cellular Component (location within cell)
Molecular Function

about 1000 categories of functions

Biological Pathways

MicroarrayDatabases
Largeexperimentsmayhavehundredsof
individualarrayhybridizations
Corelabataninstitutionormultiple
investigatorsusingonemachinedata
archiveandvalidateacrossexperiments
Datamininglookforsimilarpatternsof
geneexpressionacrossdifferentexperiments

PublicDatabases
GeneExpressiondataisanessentialaspectof
annotatingthegenome
Publicationanddataexchangeformicroarray
experiments
Datamining/Metastudies
CommondataformatXML
MIAME(MinimalInformationAbouta
MicroarrayExperiment)

GEOattheNCBI

ArrayExpressatEMBL

GeneExpression
Technologies

cDNA(EST)libraries
SAGE
Microarray
rtPCR
RNAseq

TheCancerGenomeAnatomy
Project
CGAPhascollectedalargeamountof
cDNAandrelateddataonline
http://cgap.nci.nih.gov/
cDNAlibrariesfromvarioustissues
searchforgenes
compareexpressionlevels

SAGE
SerialAnalysisofGeneExpressionisa
technologythatsequencesveryshort
fragmentsofmRNA(10or17bp)thathave
beenrandomlyligatedtogether
Theshorttagsareassignedtogenesand
thenrelativecountsforeachgeneare
computedforcDNAlibrariesfromvarious
tissues

SAGEGenie

SAGEAnatomicViewer
SAGEDigitalGeneExpressionDisplayer
DigitalNorthern
SAGEExperimentViewer

Microarray
GEOdatabaseatNCBI
Microarrayexperiments

Definedarrays
Publishedresults
Alsolotsofinconclusiveexperiments
Toolstosearchforspecificgenes
Unreliabletosearchfortissueordiseasein
experimentdescriptiontext

RNAseq
NextGenerationDNAseqencing
NYUcurrentlyhasoneIlluminaGenome
Analyser
generatesmorethan1millionRNAsequences
persample

CurrentlyseekingfundingforaRoche/454
produces100Kreadsof250400bp

CountTranscripts
Techologyexiststoaccuratelycount
transcriptsandcomparesamples
DigitalGeneExpression
Canalsoidentifyalternateisoforms,splice
variants,etc.