Question: The Easiest/Fastest Way To Get From Bam To Tpm Or Rpkm
0
gravatar for Nick
5.5 years ago by
Nick260
Spain
Nick260 wrote:

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.

rpkm tophat2 rna-seq bam bowtie2 • 7.1k views
ADD COMMENTlink modified 5.1 years ago by dfernan640 • written 5.5 years ago by Nick260
4
gravatar for Madelaine Gogol
5.5 years ago by
Madelaine Gogol5.0k
Kansas City
Madelaine Gogol5.0k wrote:

Edger rpkm() function?

ADD COMMENTlink written 5.5 years ago by Madelaine Gogol5.0k

Thanks - this would be really neat. I just checked the manual but can't quite understand how to produce it from a DGEList object. Can you, perhaps, give an example?

ADD REPLYlink written 5.5 years ago by Nick260

I also saw that logCPM (counts per million) is part of the topTags object but I don't understand how to interpret it. Is there a way to derive RPKM from it?

ADD REPLYlink written 5.5 years ago by Nick260
1

Did you try ?rpkm

rpkm(x, gene.length, normalized.lib.sizes=TRUE, log=FALSE, prior.count=0.25)

Where x is a DGElist

ADD REPLYlink modified 5.5 years ago • written 5.5 years ago by Madelaine Gogol5.0k
2
gravatar for dfernan
5.1 years ago by
dfernan640
United States
dfernan640 wrote:

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

ADD COMMENTlink modified 5.1 years ago • written 5.1 years ago by dfernan640
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