Hi all,

I have a general question about normalisation.

A collaborating group has performed RNA-seq on 15 samples - three different groups, 5 samples each. Now they realised that they are only interested in a two group comparison though and they told me to forget about the third group. My question now is the following: should I normalise (I am using TMM) only the samples from the 2 groups I want to compare or still all of them together since they were measured together and belonged to the original experimental design? 

I am grateful for advice. 

Thank you!
Julia

Leaving in the 3rd group should make barely any difference in the normalization step. In the absence of large global changes in expression, normalization relationships between samples should be approximately transitive, so adding a third group should not substantially change the relative normalization of the samples in the first 2 groups. You can verify that this is the case by computing the normalization factors both ways and then comparing them. (Remember that the absolute values may change, but the relative ratios should not change much.)

In general, it is recommended to use all the available samples from an experiment in the analysis, even if you are not interested in differential expression for some of those samples. The main reason for this is that having more samples allows more accurate estimation of the gene dispersion values.