RNA-seq - Bioinformatics differential gene expressi… · RNA-seq Quantification Harm Nijveen...

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RNA-seq Quantification Harm Nijveen

Transcript of RNA-seq - Bioinformatics differential gene expressi… · RNA-seq Quantification Harm Nijveen...

Page 1: RNA-seq - Bioinformatics differential gene expressi… · RNA-seq Quantification Harm Nijveen Differential expression Which genes are higher/lower expressed between tissues, after

RNA-seq

Quantification

HarmNijveen

Page 2: RNA-seq - Bioinformatics differential gene expressi… · RNA-seq Quantification Harm Nijveen Differential expression Which genes are higher/lower expressed between tissues, after

Differentialexpression

Whichgenesarehigher/lowerexpressedbetweentissues,aftertreatment,etc.?DifferentiallyExpressedgenes(DEGs)haveanexpressionlevelthatissignificantlydifferentbetweendifferentconditions.

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RootLeaf

ExpressiongeneX

IstheexpressionofgeneXdifferentbetweenrootandleaf?

Page 4: RNA-seq - Bioinformatics differential gene expressi… · RNA-seq Quantification Harm Nijveen Differential expression Which genes are higher/lower expressed between tissues, after

ExpressiongeneX

IstheexpressionofgeneXdifferentbetweenrootandleaf?

Basedononesample:perhaps…

RootLeaf

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IstheexpressionofgeneXdifferentbetweenrootandleaf?

Basedonthesesamples:NO!

ExpressiongeneX

RootLeaf

Page 6: RNA-seq - Bioinformatics differential gene expressi… · RNA-seq Quantification Harm Nijveen Differential expression Which genes are higher/lower expressed between tissues, after

IstheexpressionofgeneXdifferentbetweenrootandleaf?

Basedonthesesamples:YES!

ExpressiongeneX

RootLeaf

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Isagenedifferentiallyexpressed?

Withonlyonemeasurement:impossibletosayWehavetoknowthewithin-treatmentvariation

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Determiningexpressionvariation

Accuratelydeterminingthevariationrequiresmanybiologicalsamples(replicates)Unfortunatelyinmostcaseweonlyhavetwoorthreereplicates

Variationhastobeestimated

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Readcountdistribution

Poissondistribution:variance=meanHoldsfortechnicalreplicates

Negativebinomial:variance>meanBetterfitforbiologicalreplicates

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https://intro-prog-bioinfo-2012.wikispaces.com/

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Variancedependsonthemean

Trapnelletal.2012

Mainassumption:Variancedependsonthemean.Objective:Findafunctionthatbestdescribestherelationshipbetweenthemeanandvariance.

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p-value

Tofinddifferentiallyexpressedgeneswecandoastatisticaltestanddetermineap-value.p-value=0.05meansthatthereisa5%chanceforanot-differentiallyexpressedgenetoshowthesekindofexpressiondifferencesBut:with10,000genesi.e.10,000tests,youcanexpect0.05x10,000=500falsepositives!

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MultipletestingcorrectionWeneedtocorrectthep-valuefordoingalargenumberoftestsWecanusedtheFalseDiscoveryRate(FDR)thatproducesanadjustedp-valuecalledq-valueq-value=0.05meansthatthereisa5%chancethattheseexpressionvaluesarefromanotdifferentiallyexpressedgene

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Sometools..

DESeq/DESeq2EdgeRSleuth(kallisto)HISAT2/StringTie/Ballgown(canquantifyisoforms)

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PlottingDEGsVolcanoplot

x:log2(foldchange)y:-log10(p-value)

MAplot

x:meanexpressiony:log2(foldchange)

AdotrepresentsonegeneReddotsaresignificant

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VolcanoplotVolcanoplot

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MAplot

Reddotshavep-adj<0.01

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Schurch,N.J.,P.Schofield,M.Gierliński,C.Cole,A.Sherstnev,V.Singh,N.Wrobel,K.Gharbi,G.G.SimpsonandT.Owen-Hughes(2015)."EvaluationoftoolsfordifferentialgeneexpressionanalysisbyRNA-seqona48biologicalreplicateexperiment."arXivpreprintarXiv:1505.02017.

Log2(|Foldchange|)

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Whicharetheinterestinggenes?

Highestfoldchange?Lowestp-value?Other?

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Comparingmultipletreatments

Timeseries,multipletissues,etc.LookforgeneswithasimilarexpressionpatternUsingvariouskindsofclusteringmethods

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Co-expression

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Andnow?

Howdothe‘usualsuspects’behave?Whichbiologicalprocessesareenriched?Whichpathwaysareenriched?Dependsonthebiologicalquestion!Continuedthisafternoon…