Oligos for COI data

[jmquestel]

I am working with paired-end COI MiSeq data and need to remove the primers. My oligos file is as follows:

primer GGWACWGGWTGAACWGTWTAYCCYCC TAIACYTCIGGRTGICCRAARAAYCA

and I am using the oligos in the make.contigs command with a pdiffs=2

mothur > make.contigs(file=current, insert=30, oligos=COI.oligos, pdiffs=2)

When I run this command a large percentage of sequences are scrapped. With reading through the SOP and some threads on here, it seems that the newer versions (I’m using 1.44.3) only allow for a max of 2 differences between both the forwards and reverse primers or else it is scrapped where older versions allowed for max of 4 differences. Is there a way to allow for a larger difference or is there a way to trim the first 26 and last 26 bp from each read before moving on with the dataset? My concern is that many usable sequences are being scrapped because there are more than two differences between the read and the primer set as COI is a highly variable gene.

[westcott]

Use make.contigs to assemble the reads, and then run pcr.seqs to remove the primers. The pcr.seqs command has parameters for the forward and reverse diffs.

mothur > make.contigs(file=current, insert=30) - assemble reads
mothur > pcr.seqs(fasta=current, pdiffs=2, rdiffs=2, oligos=COI.oligos) - remove primers
mothur > list.seqs(fasta=current) - list reads with primers successfully removed
mothur > get.seqs(group=current, accnos=current) - select “good” reads from group file created by make.contigs.