Filter Feature Table

Sounds like you are following a qiime1-style OTU filtering protocol.

I have some changes to recommend.

If you used dada2 or deblur to denoise your sequences, then abundance-based filtering should not be necessary (though it does not hurt, either, if you want to remove low-abundance features that slow down downstream analysis steps).

Do not rarefy your table, unless if you have good reason to do so. In QIIME2, all necessary normalization steps are largely built in to different actions, e.g., rarefying is built in to the diversity core-metrics pipeline prior to alpha and beta diversity analyses. Other downstream steps like gneiss and ancom have their own normalization built in and rarefying should NOT be performed prior to any type of differential abundance analysis.

The point of rarefying — when it is performed — is to control for uneven sampling depth. So if you are using rarefying to normalize prior to, e.g., alpha diversity, then yes you should rarefy again! Do not rarefy, then filter, then rarefy again. Just filter however you need, then rarefy immediately prior to whatever analysis needs it. Don't use that rarefied table for other analyses.

I hope that helps!

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