Hello, I'd like to make an open question for anyone who can help us

We are doing several RNA-seq analysis with Deseq2. Our purpose is to increase the n of our study (as far as we sequence more samples or include new datasets), but still stablish the same comparations even if increasing the number of groups. For making the question easier of understanding I'll put two examples. In the first analysis we just analyze two cathegories, for example extreme ages (le't say young and old) and we obtain several P-adjusted values at the end of the analysis with a specific number of missregulated genes.

On the other hand, when we increase the number of categories on the analysis (for example, intermediate ranges of ages or different diet conditions), but we still want to perform the same comparison of the same samples than before (young and old), we obtain different p-adjusted values and therefore different missregulated genes, even with the same samples.

We supose that this is due to the statistic test and multiple comparisons, but we should obtain similar results comparing the same samples (otherwise, it will be a problem for comparing results, reproducibility, stablishment of signatures, increasing the n and so on) but we are not sure about how to proceed or if anyone had this problem before and what is the standart.

Any idea about where is the problem or how can we solve it?

Thank you so much in advance!!

Thank you very much for your help Michael!