DNA Microarray Data Analysis

162 Pages·2003·12.908 MB·English

by  Tuimala J., Laine M.M.

DNA Microarray
Data Analysis
TOMIPASANEN, JANNASAARELA, ILANASAARIKKO, TEEMUTOIVANEN,
MARTTITOLVANEN, MAUNOVIHINENANDGARRYWONG
EDITORS JARNOTUIMALA AND M. MINNALAINE
CSC
DNA Microarray Data Analysis

DNA Microarray Data Analysis
Editors
JarnoTuimala
M.MinnaLaine
CSC,theFinnishITcenterforScience
CSC – Scientific Computing Ltd. is a non-profit organization for high-
performancecomputingandnetworkinginFinland. CSCisownedbythe
MinistryofEducation. CSCrunsanationallarge-scalefacilityforcompu-
tationalscienceandengineeringandsupportstheuniversityandresearch
community. CSCisalsoresponsiblefortheoperationsoftheFinnishUni-
versityandResearchNetwork(Funet).
All rights reserved. The PDF version of this book or parts of it can be
usedinFinnishuniversitiesascoursematerial,providedthatthiscopyright
noticeisincluded. However,thispublicationmaynotbesoldorincluded
aspartofotherpublicationswithoutpermissionofthepublisher.
(cid:1)c Theauthorsand
CSC–ScientificComputingLtd.
2003
ISBN952-9821-89-1
http://www.csc.fi/oppaat/siru/
Printedat
PicasetOy
Helsinki2003
DNAmicroarraydataanalysis 5
Preface
ThisisthefirsteditionoftheDNAmicroarraydataanalysisguidebook. Although
inventedinthemid-90s,DNAmicroarraysarestillnoveltiesasbiomedicalresearch
tools. DNA microarraysgeneratelargeamountsofnumericaldata, whichshould
beanalyzedeffectively.
In this book, we hope to offer a broad view of basic theory and techniques
behindtheDNAmicroarraydataanalysis. Ouraimwasnottobecomprehensive,
butrathertocoverthebasics, whichareunlikelytochangemuchoveryears. We
hope that especially researchers starting their data analysis can benefit from the
book.
The text emphasizes gene expression analysis. Topics, such as genotyping,
arediscussedshortly.Thisbookdoesnotcoverthewet-labpractises,suchassam-
plepreparationorhybridization. Rather,westartwhenthemicroarrayshavebeen
scanned,andtheresultingimagesanalyzed. Inotherwords,wetakethefileswith
signalintensities,whichusuallygeneratequestionssuchas: “Howisthedatanor-
malized?” or“HowdoIidentifythegeneswhichareupregulated?”. Weprovide
somesimplesolutionstothesespecificquestionsandmanyothers.
Each chapter has a section on suggested reading, which introduces some of
therelevantliterature. Severalchaptersalsoincludedataanalysisexamplesusing
GeneSpringsoftware.
This edition of the book was written by M. Minna Laine (chapters 4, 8 and
14),TomiPasanen(chapter11),JannaSaarela(chapters2and3), IlanaSaarikko
(chapter8),TeemuToivanen(chapter14),MarttiTolvanen(chapter12),JarnoTu-
imala(chapters4,6,7,8,9,10,13and15),MaunoVihinen(chapters10,11and
12),andGarryWong(chapters1and5).
Juha Haataja and Leena Jukka are warmly acknowledged for their support
duringtheproductionofthisbook.
Weareveryinterestedinreceivingfeedbackaboutthispublication.Especially,
ifyoufeelthatsomeessentialtechniquehasbeenmissed,letusknow.Pleasesend
yourcommentstothee-mailaddressJarno.Tuimala@csc.fi.
Espoo,19thMay2003
Theauthors
6 DNAmicroarraydataanalysis
List of Contributors
M.MinnaLaine MarttiTolvanen
CSC,theFinnishITcenterforScience InstituteofMedicalTechnology
Tekniikantie15aD Lenkkeilijänkatu8
02101Espoo 33520Tampere
Finland Finland
TomiPasanen JarnoTuimala
InstituteofMedicalTechnology CSC,theFinnishITcenterforScience
Lenkkeilijänkatu8 Tekniikantie15aD
33520Tampere 02101Espoo
Finland Finland
JannaSaarela MaunoVihinen
BiomedicumBiochipCenter InstituteofMedicalTechnology
Haartmaninkatu8 Lenkkeilijänkatu8
00290Helsinki 33520Tampere
Finland Finland
IlanaSaarikko GarryWong
CentreforBiotechnology A.I.Virtanen-institute
Tykistökatu6 UniversityofKuopio
20521Turku 70211Kuopio
Finland Finland
TeemuToivanen
CentreforBiotechnology
Tykistökatu6
20521Turku
Finland
Contents 7
Contents
Preface 5
ListofContributors 6
I Introduction 14
1 Introduction 15
1.1 Whyperformmicroarrayexperiments? . . . . . . . . . . . . . 15
1.2 Whatisamicroarray? . . . . . . . . . . . . . . . . . . . . . . 15
1.3 Microarrayproduction . . . . . . . . . . . . . . . . . . . . . 16
1.4 WherecanIobtainmicroarrays? . . . . . . . . . . . . . . . . 17
1.5 ExtractingandlabelingtheRNAsample . . . . . . . . . . . . 19
1.6 RNAextractionfromscarsetissuesamples . . . . . . . . . . . 19
1.7 Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.8 Scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.9 Typicalresearchapplicationsofmicroarrays . . . . . . . . . . 21
1.10 Experimentaldesignandcontrols . . . . . . . . . . . . . . . . 22
1.11 Suggestedreading . . . . . . . . . . . . . . . . . . . . . . . . 23
2 AffymetrixGenechipsystem 25
2.1 Affymetrixtechnology . . . . . . . . . . . . . . . . . . . . . 25
2.2 SingleArrayanalysis . . . . . . . . . . . . . . . . . . . . . . 25
2.3 Detection p-value . . . . . . . . . . . . . . . . . . . . . . . . 26
2.4 Detectioncall . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.5 Signalalgorithm . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.6 Analysistips . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.7 Comparisonanalysis . . . . . . . . . . . . . . . . . . . . . . 27
2.8 Normalization . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.9 Change p-value . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.10 Changecall . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.11 SignalLogRatioAlgorithm . . . . . . . . . . . . . . . . . . . 29
3 Genotypingsystems 31
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
8 DNAmicroarraydataanalysis
3.2 Methodologies . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.3 Genotypecalls . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.4 Suggestedreading . . . . . . . . . . . . . . . . . . . . . . . . 33
4 Overviewofdataanalysis 34
4.1 cDNAmicroarraydataanalysis . . . . . . . . . . . . . . . . . 34
4.2 Affymetrixdataanalysis . . . . . . . . . . . . . . . . . . . . 35
4.3 Dataanalysispipeline . . . . . . . . . . . . . . . . . . . . . . 35
5 Experimentaldesign 38
5.1 Whydoweneedtoconsiderexperimentaldesign? . . . . . . . 38
5.2 Choosingandusingcontrols . . . . . . . . . . . . . . . . . . 38
5.3 Choosingandusingreplicates . . . . . . . . . . . . . . . . . . 39
5.4 Choosingatechnologyplatform . . . . . . . . . . . . . . . . 39
5.5 Geneclusteringv.geneclassification . . . . . . . . . . . . . . 40
5.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
5.7 Suggestedreading . . . . . . . . . . . . . . . . . . . . . . . . 41
6 Basicstatistics 42
6.1 Whystatisticsareneeded . . . . . . . . . . . . . . . . . . . . 42
6.2 Basicconcepts . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.2.1 Variables . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.2.2 Constants . . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.2.3 Distribution . . . . . . . . . . . . . . . . . . . . . . . . . 42
6.2.4 Errors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.3 Simplestatistics . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.3.1 Numberofsubjects . . . . . . . . . . . . . . . . . . . . . 43
6.3.2 Mean(m) . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.3.3 Trimmedmean . . . . . . . . . . . . . . . . . . . . . . . 43
6.3.4 Median . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
6.3.5 Percentile . . . . . . . . . . . . . . . . . . . . . . . . . . 44
6.3.6 Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
6.3.7 Varianceandthestandarddeviation . . . . . . . . . . . . 44
6.3.8 Coefficientofvariation . . . . . . . . . . . . . . . . . . . 44
6.4 Effectstatistics . . . . . . . . . . . . . . . . . . . . . . . . . 44
6.4.1 Scatterplot . . . . . . . . . . . . . . . . . . . . . . . . . 44
6.4.2 Correlation(r) . . . . . . . . . . . . . . . . . . . . . . . 45
6.4.3 Linearregression . . . . . . . . . . . . . . . . . . . . . . 46
6.5 Frequencydistributions . . . . . . . . . . . . . . . . . . . . . 47
6.5.1 Normaldistribution . . . . . . . . . . . . . . . . . . . . . 47
6.5.2 t-distribution . . . . . . . . . . . . . . . . . . . . . . . . 49
6.5.3 Skeweddistribution . . . . . . . . . . . . . . . . . . . . . 49
6.5.4 Checkingthedistributionofthedata . . . . . . . . . . . . 50
Contents 9
6.6 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . 51
6.6.1 Log -transformation . . . . . . . . . . . . . . . . . . . . 52
2
6.7 Outliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
6.8 Missingvaluesandimputation . . . . . . . . . . . . . . . . . 53
6.9 Statisticaltesting . . . . . . . . . . . . . . . . . . . . . . . . 54
6.9.1 Basicsofstatisticaltesting . . . . . . . . . . . . . . . . . 54
6.9.2 Choosingatest . . . . . . . . . . . . . . . . . . . . . . . 55
6.9.3 Thresholdfor p-value . . . . . . . . . . . . . . . . . . . 55
6.9.4 Hypothesispair . . . . . . . . . . . . . . . . . . . . . . . 55
6.9.5 Calculationofteststatisticanddegreesoffreedom . . . . 56
6.9.6 Criticalvaluestable . . . . . . . . . . . . . . . . . . . . . 57
6.9.7 Drawingconclusions . . . . . . . . . . . . . . . . . . . . 57
6.9.8 Multipletesting . . . . . . . . . . . . . . . . . . . . . . . 57
6.10 Analysisofvariance . . . . . . . . . . . . . . . . . . . . . . . 58
6.10.1 BasicsofANOVA . . . . . . . . . . . . . . . . . . . . . 58
6.10.2 Completelyrandomizedexperiment . . . . . . . . . . . . 58
6.11 StatisticsusingGeneSpring . . . . . . . . . . . . . . . . . . . 60
6.11.1 Simplestatistics . . . . . . . . . . . . . . . . . . . . . . 60
6.11.2 Tranformations . . . . . . . . . . . . . . . . . . . . . . . 60
6.11.3 Scatterplotandhistogram . . . . . . . . . . . . . . . . . 60
6.11.4 Correlation . . . . . . . . . . . . . . . . . . . . . . . . . 61
6.11.5 Linearregression . . . . . . . . . . . . . . . . . . . . . . 61
6.11.6 One-samplet-test . . . . . . . . . . . . . . . . . . . . . . 62
6.11.7 Independentsamplest-testandANOVA . . . . . . . . . . 62
6.12 Suggestedreading . . . . . . . . . . . . . . . . . . . . . . . . 64
II Analysis 65
7 Preprocessingofdata 66
7.1 Rationaleforpreprocessing . . . . . . . . . . . . . . . . . . . 66
7.2 Missingvalues . . . . . . . . . . . . . . . . . . . . . . . . . . 66
7.3 Checkingthebackgroundreading . . . . . . . . . . . . . . . . 68
7.4 Calculationofexpressionchange . . . . . . . . . . . . . . . . 69
7.4.1 Intensityratio . . . . . . . . . . . . . . . . . . . . . . . . 69
7.4.2 Logratio . . . . . . . . . . . . . . . . . . . . . . . . . . 70
7.4.3 Foldchange . . . . . . . . . . . . . . . . . . . . . . . . . 71
7.5 Handlingofreplicates . . . . . . . . . . . . . . . . . . . . . . 71
7.5.1 Typesofreplicates . . . . . . . . . . . . . . . . . . . . . 71
7.5.2 Timeseries . . . . . . . . . . . . . . . . . . . . . . . . . 71
7.5.3 Case-controlstudies . . . . . . . . . . . . . . . . . . . . 72
7.5.4 Poweranalysis . . . . . . . . . . . . . . . . . . . . . . . 72
7.5.5 Averagingreplicates . . . . . . . . . . . . . . . . . . . . 72
7.6 Checkingthequalityofreplicates . . . . . . . . . . . . . . . . 72