I want to follow up on one user’s comment: "You don’t need any singletons to calculate alpha-diversity metrics like Shannon/Simpson. " from https://forum.qiime2.org/t/singletons-and-diversity-richness-indices/2971
In the formula of Shannon index (https://en.wikipedia.org/wiki/Diversity_index#Shannon_index), singletons are considered. Does QIIME use singletons or not?
What Ben means is that the equation does not explicitly handle singletons in the same way that an alpha diversity metric like chao1 or ACE does; those metrics use singletons to estimate the number of unobserved species. Yes, singletons are considered in Shannon H, but only in the same way all other sequences are, not as an independent term used to estimate unobserved species. So Chao1 and ACE are invalid to use on filtered sequence data, but Shannon and Simpson are still valid.
QIIME 2 does whatever you tell it to do. If you filter out singletons, then no. But if you do not filter, then it can.