[BioC] DESeq and transcript-wise analysis

[Martin Morgan]

On 02/08/2012 03:41 PM, Abhishek Pratap wrote:
> 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 GenomicRanges::summarizeOverlaps for HTSeq-like counting; 
annotations might come from knownGenes (e.g., the TxDb.* annotation 
packages, or makeTranscriptDbFrom* in GenomicFeatures) or gff / friends 
via rtracklayer.

Martin

>
> 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
>>
>> _______________________________________________
>> Bioconductor mailing list
>> Bioconductor at r-project.org
>> https://stat.ethz.ch/mailman/listinfo/bioconductor
>> Search the archives:
>> http://news.gmane.org/gmane.science.biology.informatics.conductor
>
> _______________________________________________
> Bioconductor mailing list
> Bioconductor at r-project.org
> https://stat.ethz.ch/mailman/listinfo/bioconductor
> Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor


-- 
Computational Biology
Fred Hutchinson Cancer Research Center
1100 Fairview Ave. N. PO Box 19024 Seattle, WA 98109

Location: M1-B861
Telephone: 206 667-2793