[BioC] DESeq and transcript-wise analysis

[Abhishek Pratap]

Hi Elena

Good timing with me on this. I recently was contemplating the best way
to move forward for a similar analysis. HTSeq  a python based toolkit
by Simon can help you do the counting.  FYI : It can also take strand
info into account.  If you dont have stranded data you could also look
at easyrnaseq package.

So if you have an annotation file like gff/gtf with the isoform
information you could then do the read counting at isoform or gene
level based on which attribute of the gff file you select to do the
counting. Check out
http://www-huber.embl.de/users/anders/HTSeq/doc/count.html.

Also you want to keep in mind that at isoform level you would be
double counting the reads in exons which are shared in the isoforms
which can bias your results to some extent. But as Wolfgang pointed
out in a recent post if you use FDR, it should not matter a lost as
the bias will be cancelled between denominator /numerator.

You also might want to check the DEXSeq which can help infer
differential expression from RNA-Seq exons which could then be related
back to genes/isoforms.

Hope this helps and let us know about your progress. I would be
interested in learning from your experience too.

Cheers!
-Abhi

----------------------------------
Abhishek Pratap
Bioinformatics Systems Analyst - 3
DOE- Joint Genome Institute
Lawrence Berkeley National Lab




On Wed, Feb 8, 2012 at 3:26 PM, Elena Sorokin <sorokin at wisc.edu> wrote:
> Greetings all,
>
> After re-reading related posts in the listserv archive, I still didn't know
> the exact answer to my question, so here goes. I'd like to use DESeq to
> measure differential isoform expression. Has Simon or anybody else written a
> script that will convert aligned reads (.bam/.sam file) into a table of
> isoform counts, suitable for input to DESEq - similar to what Simon has done
> at the gene-wise level, but instead for making a table of counts by isoform?
>
> I would try to do this myself, but I'm a novice at programming. Sorry if
> this has been answered elsewhere... If so, please let me know the link.
>
> Thanks,
> Elena
>
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