RNASeq Differential Gene Expression with two-sample Mann-Whitney test on FPKM?

[ndovu9]

I am interested in performing differential gene expression analysis of RNASeq data in two conditions with a number of biological replicates for each condition. I have obtained gene-level FPKMs for each sample. As gene-level FPKMs should be properly normalized, comparing the expression of a single gene using FPKM across multiple samples should be reasonable. Thus, my simple strategy is this:
use a two-sample, unpaired Mann-Whitney (Wilcox) test on the FPKMs for each gene in the two groups and correct for multiple hypothesis testing.

I have a few questions regarding this approach. First, is it valid? Second, would this approach be valid for other RNASeq normalization methods (in particular Transcripts per Million (TPM) generated by RSEM)? Third, how is this approach better or worse than either count-based methods (e.g. DEGSeq/edgeR) or other commonly used methods (e.g. Cuffdiff)?

[dpryan]

I guess it depends on how many samples you have and if the fpkm normalization works out OK or not. FPKM is effectively a tweaked library size-based normalization, with the accompanying issues. That aside, if you have a good number of samples such that you can properly use a Wilcoxon, then it's probably OK. Then biggest benefit to DESeq/edgeR/etc. is the information sharing between genes for dispersion estimation. If you really do have a lot of samples, though, I would suspect that that's not very important.

[sindrle]

"good number of samples"

How many is that? Approximatly?

[dpryan]

More than a few and less than a sh!t-ton*.

For real numbers you'd have to either look that up or do a quick monte-carlo (the latter is probably more useful since you would just plug in observed distributions). My pulled-from-the-hind-quarters guesstimate would be maybe 10 samples per group, since that'd be enough to get a decent sense of the distribution (but for the love of <insert random diety>, don't base anything important on that!). A quick search of the literature reveals some more modest suggestions (around 10 minimum in one group and 4 minimum in the other), though I'd have to read further to find out how robust that really is.

*interestingly, the forum replaces curse words with asterisks.

[sindrle]

Thank!
I was going to ask if 13 is ok, and I guess it is!

I would also guess 5-10 i each group..