I'm using GSVA to get an overall activity/expression pattern of various gene sets using RNA-seq data. This is generally OK, but in my case, about half of the gene sets have only two genes, and I definitely don't want to discard them. As the GSVA help documentation recommends a minimum of 5 genes in the set, I was wondering if I'm getting useful/meaningful results at all for these very small gene sets. What do you think? If GSVA is not good in this case, are there any alternative methods that might be useful or should I just go with a summary of normalized TPM values for example?

Thanks for any suggestion!

hi Endre!!! :)

the problem with two genes is that the summary measure of expression is not going to be very robust with respect to the function you are representing with these two genes. so, you can go on and do the calculations but be cautions when interpreting the results. try to also to do some graphical exploration of the gsva scores to check whether they are sensible with respect to the genes inside the gene set.

cheers,

robert.