[BioC] easyRNAseq question

[Wolfgang Huber]

Dear Nirmala

It seems that the attachent did not come through the mailing list 
server. Can you use a public (picture) server for posting the plot? And 
provide a reproducible code example.

Also, could you be more clear about what you mean by "the QQ-plot is way 
above the expected"?

	Thanks and best wishes
	Wolfgang


Jul/2/12 11:10 PM, Akula, Nirmala (NIH/NIMH) [C] scripsit::
> Thank you Simon. I tried Ensemble GTF file with HTSeq and got ~50,000 genes for testing by DESeq. We filtered the genes with low counts and the resulting file had ~23,000 genes. The problem now is the QQ-plot is way above the expected. Please see the attachment.
>
> Analysis pipeline: Tophat-HTSeq-DESeq
>
> Any suggestions will be greatly helpful.
>
> Thank you very  much.
>
> Regards,
> Nirmala
>
> -----Original Message-----
> From: Simon Anders [mailto:anders at embl.de]
> Sent: Thursday, May 31, 2012 2:31 AM
> To: bioconductor at r-project.org
> Subject: Re: [BioC] easyRNAseq question
>
> Dear Nirmala
>
> On 2012-05-27 02:25, Akula, Nirmala (NIH/NIMH) [C] wrote:
>> I used HTSeq (similar to your geneModel method) which takes the counts
>> of disjoint exons for the genes. The problem with this method is that
>> too many reads are assigned to ambiguous category and sometimes total
>> number of reads that fall on disjoint exons are too few to get a valid
>> DESeq result. Using RefSeq genes the total number of genes counted by
>> HTSeq on my data is ~14000 whereas using the bestExon method we get
>> ~22000. Do you observe similar counts with your data?
>
> It does not quite make sense that counting only for the best exons gives you more counts than counting for all exons.
>
> Could it be that the issue with UCSC GTF files described here is the source of your problems:
>
> https://stat.ethz.ch/pipermail/bioconductor/2012-April/044717.html
>
>     Simon
>
> _______________________________________________
> 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
>


-- 
Best wishes
	Wolfgang

Wolfgang Huber
EMBL
http://www.embl.de/research/units/genome_biology/huber