[BioC] DESeq and transcript-wise analysis

[Nicolas Delhomme]

Hi Thomas,

On 9 Feb 2012, at 17:21, Thomas Girke wrote:

> This study contains some strand specific RNA-Seq data:
> http://www.hubmed.org/display.cgi?uids=18423832
> 

Thanks for the pointer.

> I would expect that most RNA-Seq experiments in the near future may be
> performed in a strand-specific manner, since the strand information
> carries a lot of biologically relevant information in this application
> domain. Thus, adding analysis support for it is definitely not a waste
> of time.

Clearly. I would have already done had I had the time.

> 
> I have not used easyRNASeq yet, but I will certainly give it a try.

Let me know when you do.

> 
> In my group we currently use for RNA-Seq analysis the following
> components: Rsubread (or tophat) -> rtracklayer/Rsamtools/GenomicRanges
> -> DESeq/edgeR. This allows any type strand and non-strand specific read
> counts for exons, transcripts, genes, intergenic features, etc. A huge
> advantage of this environment is its flexibility and broad application
> spectrum for most applications domains in the NGS field, such as
> SNP-Seq, ChiP-Seq, smallRNA-Seq, etc. For instance, our ChIP-Seq
> analysis routines use most of these tools plus some peak callers.

Exactly why I have been developing and using easyRNASeq as well.

Cheers,

Nico

> 
> Thomas
> 
> On Thu, Feb 09, 2012 at 01:38:02PM +0000, Nicolas Delhomme wrote:
>> Dear Abhi,
>> 
>> If you could point me to some published strand specific data or let me get an excerpt of yours, I could easily had strand-specificity in the easyRNASeq package.
>> 
>> Thanks,
>> 
>> Nico
>> 
>> ---------------------------------------------------------------
>> Nicolas Delhomme
>> 
>> Genome Biology Computational Support
>> 
>> European Molecular Biology Laboratory
>> 
>> Tel: +49 6221 387 8310
>> Email: nicolas.delhomme at embl.de
>> Meyerhofstrasse 1 - Postfach 10.2209
>> 69102 Heidelberg, Germany
>> ---------------------------------------------------------------
>> 
>> 
>> 
>> 
>> 
>> On 9 Feb 2012, at 00:41, 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 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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