I want to analyse a MiSeq run using mothur. Our PCR libraries were generated with barcoded primers without adapters. Due to the separate adapter ligation, read 1 and 2 did not correspond to the forward or reverse primer. Is it possible to process these “unsorted” pairs of reads with the make.contigs command?
Or is there another possibility to sort (flip) the sequences before or after the alignment?
I’m not sure how the sequences would come off the machine “unsorted” since the same cluster is sequenced in each read and outputted in the same order. Do you perhaps mean that for some clusters the sequence is in the forward orientation and in some they’re in the reverse? If this is the case, then I would suggest proceeding through like normal, but when you run align.seqs use flip=T to get everything in the correct orientation. If this isn’t the case, then I’m not sure I understand what’s going on at your end…
Thanks a lot. Of course you are right. My question was about the precessing of a file with clusters of different orientation.
Using align.seqs with flip=T is a really good option. But I want to use make.contigs for trimming the primers and barcodes as well. I tried it with an example file. Unfortunately, this failed for the reverse orientated clusters. I think a workaround could be: make.contigs without oligo file, align.seqs with flip=T, trim.seqs with the oligo file and again align.seqs. Is that ok or is there a smarter solution?
In trim.seqs we have the checkorient option (http://www.mothur.org/wiki/Trim.seqs#checkorient), this may be another way of doing what you want