How to carry out differential gene expression starting with a table of TMM normalised values without access to the raw reads?
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benedict.ng ▴ 20
@benedictng-11283
Last seen 6.5 years ago

I already have a table of TMM normalised RNA-seq values, but do not have access to the raw reads. Is it possible to do an MDS plot and calculate the common dispersion, and then do a differential gene expression? I have tried using edgeR to read the normalised values into a $counts table of a DGEList, but have not had much success. Estimating the common dispersion creates an error.

 

edger raw reads TMM normalised values DGEList differential gene expression • 1.7k views
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@steve-lianoglou-2771
Last seen 4 months ago
United States

What do you mean by "TMM normalized" RNA-seq values?

Is this the equivalent of calling cpm(calcNormFactors(y)), where y would be the DGEList (with counts) object that you wish you had right now?

If this is the case, I'd log2 transform these and pass through eBayes(..., trend=TRUE).

Assuming your.data is the matrix of cpm values and you have 3 replicates per sample, that would look something like this:

## first filter your data to remove lowly expressed genes
filtered <- rowSums(your.data > 1) >= 3
dat <- log2(filtered + small.prior.count.if.you.have.0s)
fit <- eBayes(lmFit(dat, design), trend=TRUE)
res <- topTable(fit, ...)

Refer to this thread for more information:

A: What was the very first paper to describe "limma-trend"?

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Thank you very much Steve!! Will try this out. Any advice on how to generate an MDS or PCA plot? You are correct that calcNormFactor(y) was called to get my data of TMM normalised values. Thanks once again.

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It would be best to read some case studies, for example

  http://f1000research.com/articles/5-1408

or the case study in the limma User's Guide. These case studies use voom(), but most of the process remains the same with log2CPM input. These examples will tell you how to make an MDS plot and many other things.

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Thank you very much for your help Gordon!!

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You haven't actually answered Steve's question about what sort of "normalised values" you have. You should investigate what quantities you have, what the values specifically represent, because it may make a difference.

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You are absolutely correct Gordon. I will do some investigation and see what the values represent. Thank you.

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