Removing Primers with make.contigs

Commands in mothur
1

I received demultiplexed data from my sequence provider with the adaptors and indexes removed, but the primer were still attached to sequence. I would like to remove the primer sequences during the make.contigs command. I previously ran the make.contigs command using a stability.file seen below:
Outfall NI01-Outfall_S1_L001_R1_001.fastq NI01-Outfall_S1_L001_R2_001.fastq
SEDLB NI02-SEBLB_S2_L001_R1_001.fastq NI02-SEBLB_S2_L001_R2_001.fastq

The code ran successfully but my NBases was around 291-300 when it should have been ~250. I know I need to re-run the make.contigs again with an oligos file and have created seen below:

primer GTGCCAGCMGCCGCGGTAA GGACTACHVGGGTWTCTAAT
#BARCODE CGCTCATT ATAGAGGC Outfall
#BARCODE TCCGGAGA ATAGAGGC SEDLB

I have told it to ignore the barcode sequences because they are not supposed to be present.
However when I run mothur >make.contigs(file=stability.files, oligos=testoligos.txt, processors=4), I have a warning that says since I did not provide any group names in the oligos file it is unable to create a group file? I am thinking the group file is likely needed for downstream processing and to keep sequences within their sample grouping. Any advice?

2

This could get tricky… Mothur doesn’t really want you to do it that way because you technically could try to assign names in the oligos file and that would get confusing. A better option would be to assemble your reads with make.contigs and then trim the paired primer with trim.seqs().

make.contigs(file=yourFile)
trim.seqs(fasta=current, oligos=yourOligos, pdiffs=2) - won’t create a group since you don’t have any names
list.seqs(fasta=current) - list trimmed sequences
get.seqs(group=yourGroupFIle) - will make sure any sequences that were removed due to the primer are removed from the group file.