Hi, I struggeling at the moment with an 16S-rDNA amplicon analysis. First I analyzed everything on phylum level (align.seqs, classify.seqs and so on…). Everything worked fine.
However, I could classify only little in lower taxonomic levels and I wanted therefore analyst OTUs. I used the following commands in the following order:
trim.seqs (I decided to take only sequences longer then 400 bp)
unique.seqs
align.seqs
filter.seqs (I used trump=-, vertical=F)
dist.seqs
cluster
I have around 4500 Sequences and for some reason I get only unique sequences but no OTUs. Has anyone an idea what I might do different?
There could be a bunch of reasons for this. But I think it would be easier to diagnose if you were to use one of the more mainstream pipelines, unless you have a really good reason for altering it like you have. I’m assuming that you’re using either 454 or Sanger data. You’re missing a screen.seqs step to get things to overlap, your filter.seqs step doesn’t really make sense (trump=-, vertical=F should be trump=., vertical=T), you you aren’t unique/preclustering after filter, and you aren’t chimera checking. If you can fix these (see the 454 SOP) and then get back to us on how things are looking we can go from there.
Pat