We use a mixture of 4 different reverse primers for our bacterial 454 metagenomic projects. When I try using multiple reverse primers in my oligo file, mothur ends up rejecting the vast majority of my sequences (and this data set worked fine with the trimming command using RDP, which does accept multiple reverse primers). Could there be support for multiple reverse primers?
This is probably because your sequences aren’t long enough to extend all the way through the reverse primer. We typically do not include reverse primers when we run trim.seqs. You can trim the sequences with those primers by several methods…
- Develop a hard filter that masks out the reverse primer region
- Align your sequences, select a start and stop position using screen.seqs based on summary.seqs, and then run filter.seqs
The second is actually better because it allows you to make sure that your sequences overlap over the same region and so you are not comparing evolutionary apples to oranges.
Hope this helps,