[BioC] RNA-seq

[Mark Robinson]

Hi Sridhara.

I'm having trouble understanding what you have already done (perhaps show some code?) and what exactly are asking, but I'll take a stab.

When you say "the DGE plot", what are you referring to?  A plot produced by plotSmear()?  If so, then yes, the log-ratios (and 'logConc') will take into account the normalization factors.  But, this still doesn't lead to "normalized counts".  For this, you could do something like:

d <- DGEList(counts=...)
d <- calcNormFactors(d)

rpm <- t(t(d$counts) / (d$samples$lib.size*d$samples$norm.factors)) * 1e6

Here, 'rpm' means reads per million, having used what you might call 'effective' library sizes i.e. adjusted for relative composition differences.  Of course, if you are doing RNA-seq, you may want to include a term in there for gene length, which will make it more like an RPKM.  It sort of depends what your objective is here, because none of this is really necessary for differential expression analysis (as you will see from edgeR user's guide).  Note that the 'rpm' table is not to be used for any statistical analysis of differential expression, since the data has been put on an arbitrary scale.

Hope that helps.

Cheers,
Mark

On 2011-01-25, at 11:40 AM, Sridhara Gupta Kunjeti wrote:

> Hi,
>> I am a PhD candidate at University of Delaware and area of research is
>> RNA-seq and functional genomics in Phytophthora phaseoli. I was impressed by
>> your paper in Genome Biology on "A scaling normalization method for
>> differential expression analysis of RNA-seq data". I did use the edgeR guide
>> and user manual and it makes lot of sense when the DGE in a plot graph.
>> If I am correct the DGE plot (Visualising DGE results) is plotted using the
>> normalized tag counts for each gene ID, please correct me if I am wrong?
>> If so, I was wondering if I can generate a list or table or gene ID with
>> the normalized tag counts? I would appreciate if you let me know if this can
>> be done.
>> 
>> Thanks,
>> Sridhara
>> 
>> 
>> --
>> Sridhara G Kunjeti
>> PhD Candidate
>> University of Delaware
>> Department of Plant and Soil Science
>> email- sridhara at udel.edu
>> Ph: 832-566-0011
>> 
> 
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------------------------------
Mark Robinson, PhD (Melb)
Epigenetics Laboratory, Garvan
Bioinformatics Division, WEHI
e: mrobinson at wehi.edu.au
e: m.robinson at garvan.org.au
p: +61 (0)3 9345 2628
f: +61 (0)3 9347 0852
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