Hi, Dear Drs. Lianoglou and Smyth:

 

yes, I pretty found all of your referred link excpet for the forum link. The consensus is those raw_counts are not true raw counts. In fact, the V1 RNA-seq data did have a field as raw counts, but only a small portion of samples compared to the V2 RNAseq data, which is kind of dilemma. I may try limma voom and EBSeq and see. 

 

Thanks for your efforts!

best

 

Ming


 

> Date: Thu, 30 Jan 2014 05:57:06 -0800
> From: lianoglou.steve@gene.com
> To: smyth@wehi.edu.au
> CC: bioconductor@r-project.org
> Subject: Re: [BioC] questions on edgeR package
> 
> Hi,
> 
> On Thu, Jan 30, 2014 at 2:07 AM, Gordon K Smyth <smyth@wehi.edu.au> wrote:
> [snip]
> > I think the point is that Ming has already downloaded the v2 data, and the
> > so-called "raw counts" turned out not to be counts.
> 
> I see ... I wasn't paying very close attention to this thread until my
> last reply and hadn't realized that the OP has pretty much hashed
> these things out.
> 
> > If you want to dig to find out what the "raw counts" are exactly, that would
> > be a great service, because I am just guessing. The TCGA documentation just
> > says they are from RSEM.
> 
> After some digging: there are several pages that show the output of
> the rna-seq pipeline, such as this one:
> 
> https://wiki.nci.nih.gov/display/TCGA/RNASeq+Version+2
> 
> These suggest (under "File field descriptions") that there is a column
> int the gene-level summaries file called "raw_counts" which are "The
> number of reads mapping to this gene". Digging around a bit more, you
> find that this is really RSEM expected counts. This page was helpful:
> 
> https://confluence.broadinstitute.org/download/attachments/29790363/DESCRIPTION.txt?version=1&modificationDate=1363806109000
> 
> At the bottom of the page under the "Column Headers" section is where
> you get the required detail. This thread on RSEM-users mailing list
> also confirms:
> 
> https://groups.google.com/forum/#!topic/rsem-users/H1cswrvvmPs
> 
> The raw_count columns are really the expected counts from RSEM (where
> the authors of RSEM suggest that these rounded numbers are suitable
> for edgeR / DESeq ;-)
> 
> Perhaps the OP would best use EBSeq for the TCGA "raw_counts" data,
> or, as you suggest, limma::voom since these are actually the expected
> counts.
> 
> If you *really* want to use edgeR, the first link I pointed to
> suggests that the "raw_count" column in the "exon_quantification.txt"
> are actually the raw counts to the exon (can the OP confirm that they
> are actually integers?). If so, perhaps you could sum this up per gene
> and then continue. I suspect you'd likely then be double counting
> exon-spanning reads, which might be problematic. In principle, you
> could then subtract the tallies in the junction_quantification.txt to
> accommodate for that -- which all seems like a lot of work if
> limma::voom and EBSeq will work just as well on the RSEM expected
> counts.
> 
> Hope that helps,
> 
> -steve
> 
> 
> -- 
> Steve Lianoglou
> Computational Biologist
> Genentech
> 
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