Is calcuNormFactors necessary in the calculation of RPKM?
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Entering edit mode
2.3 years ago
wangdp123 ▴ 250

Hi there,

It seems that there are two ways of calculating RPKM values based on rpkm function of edgeR from the output of featurecounts. One uses calcuNormFactors and the other doesn't.

I wonder which one is more appropriate for generating RPKM values from RNA-Seq analysis? Why?

1)

x <- DGEList(counts=fc$counts, genes=fc$annotation[,c("GeneID","Length")])
x_rpkm <- rpkm(x,x$genes$Length)


2)

y <- DGEList(counts=fc$counts, genes=fc$annotation[,c("GeneID","Length")])
y <- calcNormFactors(y)
y_rpkm <- rpkm(y,y$genes$Length)


Many thanks,

Tom

RNA-Seq edgeR RPKM • 827 views
1
Entering edit mode
2.3 years ago
ATpoint 54k

It is the second one as this takes into account the TMM-derived scaling factors which, beyond sequencing depth also corrects for library composition changes. There is a video on YT on TMM normalization (StatQuest series) that explains this quite well.