reverse primer in trim.seqs oligo file

Commands in mothur
1

I’ve been trying to trim the ITS1 rDNA region out of pyrosequencing (~450bp, 454TI) reads by trimming from my forward primer (located at the 3’ end of the 18S) to a reverse primer located on the 5.8S. I’ve defined the primers in the oligo file but all reads end up in the scrap file with the flag indicating the reverse primer was not found. When using trim.seqs with just the forward primer, everything works. I’ve tried using the reverse primer in it’s complement, revcom, and reverse… nothing seems to work. The kicker is I can grep out reads from the scrap file that contain the reverse primer sequence! What gives?

oligo file:

#ITS1F
forward CTTGGTCATTTAGAGGAAGTAA
#ITS3
reverse GCATCGATGAAGAACGCAGC
#ITS3 revcom
#reverse GCTGCGTTCTTCATCGATGC
#ITS3 reversed
#reverse CGACGCAAGAAGTAGCTACG
#ITS3 complement
#reverse CGTAGCTACTTCTTGCGTCG

A single sequence from the scrap file with the ITS3 reverse primer in bold:

GJDBWV201AZ6G6|r
TTCAGGTCCTTGGTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGATCATTACAGAAAGTAAACGCGGATCAAACCGCGAACTTCTAAACCTTTGACGATTGACTCATGTTGCCTCG
GCGGGTTCTCCCGCCAGAGGATACATCAAAACTCCTGTTTTAACGGTGTTGTCTGAGCTACAAGCAACGAATCAAAACTTTCAACAACGGATCTCTTGGTTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAG
TAGTGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCCTTGGTATCCCGAGGGGCATGCCTGTTCGAGCGTCATTTCACCACTCAAGCCTGGCTTGGTGTTGGGCGACGTCCCCTTTGGGGA
CGCGCCTCGAAACGCTCGGCGGCGTGGCACCGGCTTTAAGCGTAGCAGAATCTTTCGCTTTAAAAGTCGGGGCCCCGTCTGCCGGAAGACCTACTCGCAAGGTTGACCTCGGATCAGGCAGGGATACCCGCTG

2

Sorry, but the reverse primer has to be at the end of the sequence. As an alternative, you could try aligning the sequences (muscle?) then applying a hard filter that you make to remove the reverse primer and everything that follows.

3

Bummer… These sequences are hyperdiverse across the kingdom Fungi so they won’t really align to one another with any reliability. Maybe allowing reverse primer trimming from anywhere in the sequence would be a good option for later Mothur versions? :wink:

Great program, by the way. Really makes my life a lot easier. Thanks.