Thank you!

> Date: Wed, 4 Dec 2013 15:14:06 +1100
> From: smyth@wehi.EDU.AU
> To: karenmenuz@hotmail.com
> CC: bioconductor@r-project.org
> Subject: edgeR prior.count
> 
> Dear Karen,
> 
> > Date: Mon,  2 Dec 2013 10:55:38 -0800 (PST)
> > From: "Karen [guest]" <guest@bioconductor.org>
> > To: bioconductor@r-project.org, karenmenuz@hotmail.com
> > Subject: [BioC] edgeR prior.count
> >
> >
> > I recently used the EdgeR package to analyze a RNA-Seq dataset, with 2 
> > genotypes and 3 biological replicates each.
> 
> Please update to the current Bioconductor release (edgeR 3.4.1).
> 
> > After running the exacttest, the logFC and logCPM are provided for each 
> > gene. I am a bit confused about how exactly these values are calculated.
> 
> It may be that you are expecting things to be somewhat simpler than they 
> actually are.  edgeR uses generalized linear models to compute 
> statistically efficient estimates of logCPM and logFC values.  These 
> involve an interative computation for each gene that takes into account 
> the dispersion value, library sizes and so on.  It's not just a matter of 
> computing moderated counts and then taking averages or differences.
> 
> > 1) For logCPM, I assume that this is the average expression over all 
> > samples. It is clearly not simply the averaged [counts/effective library 
> > size for each sample].
> >
> > I understand that generally speaking the original counts (or the CPM? 
> > instead) are moderated to avoid infinite values when taking logs of 
> > samples/genes with zero counts/CPM, but I'm not quite sure that I can 
> > figure out exactly how this is produced.
> 
> See ?aveLogCPM
> 
> > a) Is the same small value added to each gene for each sample or is the 
> > added value different for different genes? How is prior.count 
> > determined?
> 
> See ?predFC
> 
> As for determining the prior.count, you input the prior count yourself 
> when you run exactTest, or else the default value is used.  The 
> prior.count has no effect on the p-values.  It only affects the amount of 
> moderation applied to the reported fold changes.
> 
> > b) Are only genes that have a "0" in one sample moderated or all all 
> > genes moderated by prior.count?
> 
> See ?predFC
> 
> > c) Is there a way to see the moderated CPM for each gene and sample and 
> > not just the log (moderated CPM)?
> 
> See ?cpm
> 
> > 2) How is the logFC calculated? Is it based on moderated CPMs for each 
> > lane? Does it take the ratio of the average moderated CPM for each 
> > group?
> 
> Generalized linear model.  See ?glmFit.  Note that a generalized linear 
> model is used for the fold changes, even when using the exactTest.
> 
> Best wishes
> Gordon
> 
> > Thank you!
> >
> > -- output of sessionInfo():
> >
> > R version 3.0.1 (2013-05-16)
> > Platform: x86_64-apple-darwin10.8.0 (64-bit)
> >
> > locale:
> > [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
> >
> > attached base packages:
> > [1] stats     graphics  grDevices utils     datasets  methods   base
> >
> > other attached packages:
> > [1] edgeR_3.2.4  limma_3.16.7
> >
> > --
> > Sent via the guest posting facility at bioconductor.org.
> 
> ______________________________________________________________________
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