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Genesettes'ng RNASeqWorkshop September2016 Why? • Some'mesa=erdifferen'alexpressiontes'ng,we havealonglistof1000’sofgenes • Toodifficulttogothroughonebyone • Wanttounderstandpathwaysinvolvedinthe biologicalsystembeingstudied Genesettestsavailableinlimma • WanttotestLOTSofgenesets? – goana()func'on • GeneOntology(GO)analysis – camera()func'on • Userspecifiedgenesets • Wanttotestjustafewgenesets? – roast()func'on Basicprinciplesbehind genesettes'ng “Overlap”analysis:goana,DAVID, toppfun,GOstats(&mostweb-basedtools) 180 10 60 190genesin geneset Isanoverlapof 10significant? 70significant genes Problem:thistestisbiaseddueto thefactthatlongergenestendto havemorereadsassignedtothem GOcategorieshavedifferentavg genelengths GOseq,Youngetal,2010 Solu'on:takeintoaccountgene lengthinyourGOanalysis • goana()hastheabilitytotakeintoaccount genelengthusingthe“covariate”argument • TheGOseqbioconductorpackagecontainsthe originalmethod CAMERA • An“overlap”analysisassumesthegenesare independent • CAMERAteststherankingofthegeneset rela'vetotheothergenesintheexperiment, whiletakingintoaccountinter-gene correla'ons • Italsotakesintoaccountstrengthofevidence ofDEbyusingthemoderatedt-sta's'cs Rankgenesandmarksignature Rankgenesby differen'al expression Gene1 Gene2 Gene3 Gene4 Gene5 Gene6 Posi've signature genes Gene7 Gene8 Gene9 Nega've signature genes Gene10 Gene11 Gene12 Gene13 Gene14 Gene15 Gene16 Slidecourtesyof GordonSmyth 10 Rankgenesandmarksignature Rankgenesby differen'al expression Gene1 Gene2 Gene3 Gene4 Gene5 Gene6 Gene7 Gene8 Genome-wide barcodeplot Gene9 Gene10 Gene11 Gene12 Gene13 Gene14 Gene15 Gene16 Slidecourtesyof GordonSmyth 11 Group B -4.67 -1.34 -0.86 -0.55 -0.24 0.03 0.31 0.60 0.92 1.42 4.00 Group A 0 Enrichment 3.3 Visualisa'on: Barcodeplot+enrichmentworm statistics Slidecourtesyof GordonSmyth 12 ROASTgenesettest • Theques'onaskedis“Dothegenesinthis genesettendtobedifferen'allyexpressed?” • ItisNOTcomparedrela'vetoothergenes • Itisdesignedsuchthatif>25-50%ofgenesin thegenesetaredifferen'allyexpresseditwill besignificant • Itusessophis'catedtechniques(rota'on)to preservegene-genedependenceinthedata. Summary • Genesettes'ngtechniquesrangefromsimple (overlapanalysis)toquitecomplex(CAMERA andROAST) • Whichtestyouchoosedependsonwhatyour hypothesisis • Some'meswejustdothemall… Acknowledgements • GordonSmyth