Question: differential expression at transcript level with paired samples
gravatar for mjg
5.2 years ago by
United Kingdom
mjg20 wrote:

Dear all,

I want to do differential expression at transcript level using a rnaseq dataset that consists of paired samples (patient 1 control/treatment, patient2 control/treatment, etc.).

I have not found methods other than Ballgown and DEXSeq that are able to handle paired designs. I have also tried the option of obtaining transcript counts and use them in edgeR. However I am concerned about the fact that edgeR is not particularly adapted to do analysis on transcript counts.

Does anyone has any advice on how to go on this problem? Or if there is a way to assess my results with the latter approach I described?

Many thanks,




isoform rna-seq paired samples • 2.1k views
ADD COMMENTlink modified 5.2 years ago by Lior Pachter540 • written 5.2 years ago by mjg20

Adding on the answer of Sean Davis, both salmon and kallisto can perform bootstrapping to estimate the uncertainty of the transcript level abundances. edgeR now has functions (catchSalmon and catchKallisto) to use these bootstraps to assign an overdispersion value to each trancript. Larger mapping uncertainty means higher overdispersion, so the mapping uncertainty is taken into account during the differential analysis. Having that said, edgeR is able to perform transcript level analysis, check its manual. I think these methods were introduced to edgeR a few years ago, but not when OP asked this question.

ADD REPLYlink written 4 weeks ago by ATpoint40k
gravatar for Sean Davis
5.2 years ago by
Sean Davis26k
National Institutes of Health, Bethesda, MD
Sean Davis26k wrote:

Perhaps not the solution you were looking for, but software like kallisto and Salmon (and others) generate estimates of raw counts per transcript.  You could use those estimates in edgeR or DESeq2.  

ADD COMMENTlink written 5.2 years ago by Sean Davis26k

Thanks Sean, I will consider these on my analysis.

ADD REPLYlink written 5.2 years ago by mjg20

If you go that route, Martin Morgan has provided code to read in kallisto output:  

ADD REPLYlink modified 5.2 years ago • written 5.2 years ago by Sean Davis26k
gravatar for Lior Pachter
5.2 years ago by
Lior Pachter540
United States
Lior Pachter540 wrote:

The combination of kallisto and sleuth will allow you to do a transcript level analysis while taking into account your paired design.

ADD COMMENTlink modified 11 months ago by RamRS30k • written 5.2 years ago by Lior Pachter540
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