Download Gene set tes-ng

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