Hello. I am using the current version of mothur, and followed the exact code in the MiSeq SOP. A couple of questions.
I used trim.seqs to remove the primers after the make.contigs command - trim.seqs(fasta=stability.files.trim.contigs.unique.align, oligos=oligos.oligos, processors=4). Is this the most sensible time to remove primers, or would there be a better place for this?
Second, when I get to the stage of classify.seqs, all sequences are clustered into one group and are not assigned to each sample as outlined in stability.files. The count files do not appear to carry over the information needed to split these into the correct samples. Could you advise on this?
I used trim.seqs to remove the primers after the make.contigs command - trim.seqs(fasta=stability.files.trim.contigs.unique.align, oligos=oligos.oligos, processors=4). Is this the most sensible time to remove primers, or would there be a better place for this?
You should be able to give the oligos file to make.contigs rather than doing it in trim.seqs.
Second, when I get to the stage of classify.seqs, all sequences are clustered into one group and are not assigned to each sample as outlined in stability.files. The count files do not appear to carry over the information needed to split these into the correct samples. Could you advise on this?
I’m not sure what you are referring to as a “group” - do you mean taxonomic group or a sample? classify.seqs only really assigns sequences to taxa. You should follow the phylotype approach in the MiSeq SOP to see how to get a shared file out of taxonomic data.
For the second part of my question, when I say groups, I mean samples. I followed the MiSeq SOP on your website. The group information is not carried through the count files, which I presume should be the case instead of through names and groups files, as in previous versions?
With the newer version of mothur you should be using the count_file like we describe in the SOP. Is it possible that you left the count file out at some point in your pipeline?