Differential transcript expression / differential isoform expression tool tutorial

Question

[firestar]

I am looking for a tutorial or example workflow for differential transcript expression / differential isoform expression. I am not looking for DTU.

I just need to know if a transcript is significantly higher in condition a compared to condition b. I know it's very similar to differential gene expression using DESeq2 or edgeR for example, but I am looking for an example workflow just to make sure I am doing it correctly. I have transcript counts from salmon as the starting point. I do not have exon counts.

I have looked at IsoformSwitchAnalyzer and DRIMSeq, but those both DTU. DEXSeq seems to use exon counts and I am not sure if it's DTE. I have come across rnaseqDTU using DRIMSeq and DEXSeq but also seems to be DTU.

Answer 1

[ATpoint]

I think you want https://bioconductor.org/packages/release/bioc/vignettes/fishpond/inst/doc/swish.html which runs downstream of quantification (with bootstrap or Gibbs sampling, aks inferentisl replicates) with salmon.

[firestar]

I came across this and tried it out, but it turns out the salmon results I have were run without bootstrap replicates and it complained about it.

[ATpoint]

Then rerun it. The inferential uncertainty estimated are essential to get information on the reliability of the quantification. Transcripts per gene share most of its exonic content so many reads cannot be mapped uniquely.