calculate RPKM from featureCounts result

Question

[yuabrahamliu]

Hi all, I have a question. I got the read counts result from some microRNA-seq bam files by featureCounts. Then, I want to transform the counts to RPKM. I think maybe the rpkm() function of edgeR is helpful. However, my question is whether I need to normalize the counts result before using the rpkm() function, or just use the counts directly? Thank you so much.

Answer 1

[cindy.perscheid]

Hi yuabrahamliu,

RPKM itself is the normalization function. ;) However, I would not recommend to use RPKM for your analysis, as it is considered to be not suitable anymore (see recent discussion here and publication comparing the different normalization strategies here). I would rather recommend to use DESeq2 or TMM normalization as described in the paper.

Best,

Cindy

[yuabrahamliu]

I see. Thank you so much!