Performing differential expression study on isoforms

Question

[glady]

Hello, I have a RNAseq data(human) of 9 samples with 3 replicates each. the mapping to the reference was performed by STAR and quantification was done by RSEM. The differential expression study on genes was performed by DESeq2(taking the raw counts), but it failed for isoforms.

1) Is there some other tool with which we can perform a DE study on isoforms? 2) can we use the TPM counts instead of the raw counts for performing the DE analysis? if no. then why? 3) using tximport + deseq2 on TPM values, will this work for DE isoform study?

[Martombo]

What do you mean with DESeq2 "failed" on raw isoform counts? It sounds like the correct approach to me. RSEM does not return integer isoform counts however, there will be some rounding up to do...

[glady]

RSEM gives you two files as output - "filename.genes.results" & "filename.isoforms.results. the second file contains the expected_counts on isoforms as well as the TPM counts.

[Martombo]

These expected counts are not integers right? The only issue I see with using DESeq2 with it is to rounding them up. What else didn't work in your case?

[glady]

Yes, the counts are in integers. But if you use these counts as inputs to DESeq2, it gives you false results. Because the DESeq2 works very well for the gene counts and not for isoform counts, since the isoform are usually overlaps in the genes.

Answer 1

[Charles Warden]

To be honest, I've typically parsed the counts from Salmon / Sailfish directly (when I tried RSEM, the alignment either took a long time, or the quantification didn't seem as good: however, I assume that you have had better luck...).

However, if you are worried about bugs in your custom code (because something seems strange in DESeq2), there is a package that is supposed to help with import to DESeq2:

https://bioconductor.org/packages/release/bioc/html/tximport.html

I haven't tested it personally, but hopefully you find it useful!

It is supposed to work with RSEM output: https://bioconductor.org/packages/release/bioc/vignettes/tximport/inst/doc/tximport.html#rsem

[glady]

I didn't cam across this, it would be helpful. Thank you.

Answer 2

[Benn]

There is a chapter in the edgeR guide about differential splicing analysis (4.5). I assume that's what you want?