I just got my first Miseq dataset and I amplified the V3-V4 16S rDNA region (2x250 bp). After the clustering, i obtain a lot of OTUs (until 16.000 for one sample).
I am novice, and the question i ask myself is whether i should really after the normalization (ie the same number of sequences for all samples) if i have to remove the singletons and/or the low abundance (OTUs < 0.005%) or not. Indeed, I did the rarefaction curves without and with the removal of singletons.
When i keep the singletons, the rarefaction curves not reaching the asymptote whereas when i remove the low abundance the rarefaction curves reach the asymptote.
I wonder if that is disturbing or not reaching the asymptote ?
I have an another question, what are the calculators available to know the alpha diversity when we remove the low abundance because if i understand the chao1 estimator takes into account the singletons and doubletons and i can’t use it in this case.
Can anyone help me ?
All answers will be appreciated !