I have already aligned all my RNA-seq samples using tophat2. I've done the differential analysis using htseq-count and edgeR. I would like to get rpkm and tpm metrics. What is the easiest way to do that using the output of tophat2, htseq-count and edgeR? I also have the reference GTF. So, please, recommend a tool or a workflow that would take me to TPKM or RPKM in less than a day - I have a machine with 32GB RAM and 8 cores and my study comprises 24 samples.
2 answers
Edger rpkm() function?
Did you try ?rpkm
rpkm(x, gene.length, normalized.lib.sizes=TRUE, log=FALSE, prior.count=0.25)
Where x is a DGElist
Easiest means wrong in this case, since one cannot possibly get a right estimate for RPKMs in an RNA-Seq experiment without proper handle of multiple alignments. I'd say the easiest and still right way to do it is to use Express/Cufflinks/RSEM/etc. - they produce RPKM estimates, and RSEM produces RPKM/TPM estimates - anything else without handling of multiple alignments may be easy but it's just wrong...
You can also get RPKM estimates and transform them to TPM estimates
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