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Question: Using RNA-Seq raw count data in Weighted Gene Co-Expression Network Analysis
1
4.1 years ago by
Jon Bråte150
Norway
Jon Bråte150 wrote:

Hi,

I have gene expression count data generated by HTSeq and I wonder how I can use them in the WGCNA-package? I think one of the datasets from the turotial is: "the ratio of the mean log10 intensity (mlratio) relative to the pool derived from 150 mice". Can I use voom transformation in limma for instance?

Thanks,

Jon

modified 4.1 years ago by Steve Lianoglou12k • written 4.1 years ago by Jon Bråte150
6
4.1 years ago by
Denali
Steve Lianoglou12k wrote:

You wouldn't use a "voom transformation" ... voom doesn't perform much of a transformation at all as it simply provides something like a +0.5 smoothed logCPM estimate for the counts form its inputted DGEList (though, I will grant that this is a transformation! :-).

The magic of voom is the "sister" weights matrix that it provides, and for that to be useful,  your downstream  method would have to be one that can leverage these observational weights.

You likely want some type of "variance stabilizing transformation" of your count data, though. In the edgeR/limma world, this would involve calling cpm on your count matrix with a value somewhere between 2-5 for the "prior.count" argument (sorry, but I can't give you better guidance on the choice of "prior.count" ... picking "the right" value for that (if there can be one) seems like a bit of voodoo for the time being, but perhaps Gordon can chime in), cf:

Alternatively you could use the "varianceStabilizing" or "rlog" transformations from DESeq2, see the "Data transformations and visualization" section of the Differential analysis of count data vignette in the DESeq2 package.