Alpha diversity - rarefaction curves and Chao1

Hi mother’s users!

I’ve a simple question related with this issue (see the link): https://www.researchgate.net/post/Rarefaction_curve_and_singleton_a_unique_sequence_that_is_present_exactly_once_in_a_dataset.
What’s the meaning of doing rarefaction curves and/or estimate the species richness using Chao1 index after excluding the singletons (OTUs)?
This makes any sense to you, since both are based on singletons.
What is the meaning of estimating the true species richness in one sample/community using Chao1 after excluding the singletons (OTUs), when this index tries to inflate the number of species expected based on higher no. of singletons as well as doubletons (see the link: http://www.coloss.org/beebook/I/gut-symbionts/2/2/4).
I don’t see the point. What am I missing here?
Please, can someone shed some light here.

Thanks in advance,
@renh@

What’s the meaning of doing rarefaction curves and/or estimate the species richness using Chao1 index after excluding the singletons (OTUs)?
This makes any sense to you, since both are based on singletons.

You should not be removing singletons. Rarefaction will take care of this for you. Rarefying the Chao1 index will allow you to compare Chao1 values across samples by controlling for the uneven number of sequences found in each sample.

Pat

Thanks.
I understood your explanation.
But now, take as example this article https://www.nature.com/articles/s41598-017-04016-w. It seems that they excluded the singletons, and after that they measure alpha diversity, including both formula, Chao1 index and rarefaction curves.
If they actually did this (as it seems), what’s the reliability of the outcome of such alpha diversity parameters.

Thanks in advance,
@renh@

All I can say is that they were wrong to do this.

Pat