Question: Which software should be used to calculate the TPM values of genes?
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gravatar for wayj86
22 months ago by
wayj8610
wayj8610 wrote:

Hi all,

I am using STAR to align my reads to human genome. Then I need to calculate the TPM values of genes. There are many software that can be used to do that: Stringtie, RSEM, kallisto, Salmon, salfish, ...

So which one is best for doing that?

Many thanks, Stanley

rna-seq • 1.6k views
ADD COMMENTlink written 22 months ago by wayj8610

Do you definitively need to just calculate TPM values or do you ultimately want to perform a differential expression analysis?

If you have aligned your reads to the genome, then you should have SAM or BAM files.

You can calculate raw counts over these SAM / BAM files using featureCounts, and then read the raw counts into EdgeR or DESeq2 where you can perform normalisation.

ADD REPLYlink modified 22 months ago • written 22 months ago by Kevin Blighe52k

Thank you very much for your answer. I need TPM to perform WGCNA, actually.

ADD REPLYlink written 22 months ago by wayj8610

WGCNA can take any type of normalised and/or logged data, in fact. Were you told to just use TPM? WGCNA is fundamentally based on correlation.

ADD REPLYlink written 22 months ago by Kevin Blighe52k

Yeh. WGCNA usually takes FPKM data, but I was told TPM was much better than FPKM. That is why I need to calculate TPM.

ADD REPLYlink written 22 months ago by wayj8610

I do not believe that's true at all. WGCNA takes any data:

  • Metabolomic Z-score
  • RNA-seq normalised counts on the negative binomial scale
  • cDNA microarray log2 expression count

One could argue that TPM is better than FPKM (for differential expression analysis), but neither of these are better than TMM or the geometric median/mean methods used by EdgeR and DESeq2, respectively.

In any case, if you use featureCounts to derive raw counts, like I mentioned, you can then convert these to TPM: Calculating TPM from featureCounts output

ADD REPLYlink written 22 months ago by Kevin Blighe52k
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