Using pcr.seqs with the oligo parameter

Commands in mothur
1

Dear mothur-community,

we are at the step where we shorten our Silva-reference database to the region of interest. Here it is the V3-V4-region. Instead of using the start/end position we want to use our used primers, which are stored in the .oligo-file like following:

primer CCTACGGGNGGCWGCAG V3
primer GACTACHVGGGTATCTAATCC V4

However, we see that only the V3-primer is recognized and the reference sequences shortened at the 5’-end. Any clues why this is happening and the V4-primer is not found and trimmed?

Thanks for any advice or hint!
Regards,

2

To use the primer option you want to do something like…

primer CCTACGGGNGGCWGCAG GACTACHVGGGTATCTAATCC

Alternatively you could do…

forward CCTACGGGNGGCWGCAG V3
reverse GACTACHVGGGTATCTAATCC V4

See if either of those work.
Pat