Dear mothur creators,
I am not sure where to post this, so I will try here.
I would like your opinion on the problem (if it can be called so) that I’ve encountered.
I have ~200 samples, V4, region. Unfortunately, sequenced with 150 bp paired-end. I have always worked with 250 paired-end reads, and although make.contigs with complete overlap and a subsequent qc still resulted in lots of unique sequences for environmental and marine invertebrate microbiota, I usually ended up having reasonably few unique sequences when it comes to human gut microbiota.
This time, even after precluster, I ended up with cca 400000 unique sequences. But, what puzzles me more, is that very few of them (max 2-3%,even after fiddling with parameters to increase the sensitivity) turn out to be chimeras, regardless to the method used. I don’t say it’s wrong, but I do find it strange, as I’ ve always had considerably higher proportion of unique sequences marked as potentially chimeric.
What do you think about this? Is it normal to have so few chimeras?
Thanks in advance for your opinion,
EDIT: So I suppose the problem is simply that because with the short overlap, I am simply left with two many errors. I never remove singletons from my analysis, but do you think it is reasonable to do it this time (before chimera removal)?