Trimming reverse primers from paired end reads

Commands in mothur
1

HI,

Me and someone else from my institution have been attempting to trim the primers off our paired end sequences. Our first sequencing reads begin with our forward primer but our second R2 reads have a few (1-2) random bases before the reverse primer sequence begins.

We have tried to use the oligos option in make.contigs and also after make.contigs (without the oligos option) in trim.seqs and pcr.seqs to remove our primers but this only removes our forward primer. We have formatted the oligo file in the following manner:

primer: #forward primer# #reverse primer#

For the reverse primer we have tried it 5’ to 3’ and 3’ to 5’. We have also tried inputting its complement. In case it is due to the random nucleotides before the reverse primer we tried putting 1-2 N bases before the reverse primer in the oligo file to make the program think these were part of the primer.

We feel we have ran out of options now so we were wondering if there might be a solution?

2

Could you post a pair of reads and the barcodes and primers? It seems like we should be able to overcome this with a spacer or bdiffs and pdiffs.

3

Hi,

Sorry for the late reply. I did try using spacers but this still didn’t work. I’ve therefore started to use cutadapt instead before processing which seems to work fine.