Hi,

I’m trying to do differential abundance analysis between gut samples and mesenteric lymph nodes. Understandably, the relative abundance in lymph nodes is very low. The challenge I’m having is how to do the differential abundance analysis between those groups, given that:

- The mean library size difference between the groups is >10x. ( Lymph node= 995, gut= 70,000).
- The smallest library size is 311, the second smallest is 492.
- The sample size of the groups is small. For some groups is 4 and for some is 5.

I f I do rarefying, I would loose two samples in the group of 4, so I would have only two samples left. I’ve read in the literature than DESEQ2 is sensitive for small sample sizes. However, I don’t know how to handle this enormous differences in library sizes and if rarefying to 492 would make any sense considering the other libraries have > 60,000 reads.

Thank you very much for your help on this issue.