ANCOM tutorial: Relative frequency table error required to make PCoA plot; ANCOM on rarefied table

[Anahita_Bharadwaj]

Thanks so much for your response @llenzi. My bad... When I say "normalization", I mean "normalization by rarefaction".

@llenzi

In my view, I don’t see rarefying the data as big no-no! It is still my preferred way to get ‘quick and dirty’ diversity for a dataset (and my only option for some analysis but this is an aside … ).

ahh ok, good to know. I have done this to build some of the alpha and beta diversity metric according to the QIIME2 tutorial.
I figured if I did this, it would help me understand the differences across my samples (and different ANCOM runs) when I do pairwise ANCOM tests on them. Although the W values might be different (and therefore, not comparable), I felt that it would be more indicative of changes across my samples as they start off at the same sampling depth. Does this logic make sense at all or is the ANCOM math not setup to do this?

@llenzi

Hence, maybe trying the command above using the not-normalised abundance table may work and give the information you asking!

Good idea... I will do this and see if I get similar results.

@llenzi

To do pairwise tests, the easy way may be to select only the groups in the comparison, but still the ANCOM including all the data should give a nice overview for the main changes.

Ya, that's what I figured too. As recommended by another post, what I did was to do an "overall" ANCOM on a phylum level and then selected the phyla showing significant changes. I then filtered these phyla from the rarefied table and performed ANCOM on them (on a genus level) to parse out where the main differences are coming from. Is this a reasonable assumption to make?

Sorry, I only ask because I want to be sure I am doing it correctly. Thank you for all the help and advice. This forum is amazing!

PS: I am editing the title to include this discussion.