Trim.seqs with reverse MiSeq PE reads

Commands in mothur
1

using Mothur v.1.32.1
I am trying to use trim.seqs to pull out barcoded samples within a larger dataset and ultimately I would like to use make.contigs on those samples. I used the following trim.seqs command and example oligos file on the forward fasta and qual files and it worked beautifully, but I am having difficulties getting it to work with the reverse files.

trim.seqs(fasta=Forward.fasta, qfile=Forward.qual, oligos=barcode.txt, allfiles=t)

barcode AAGCCTTGCGTCAGACA Sample 1
barcode AAGCCAAGGGAGGAGAC Sample 2

In my reverse files, the barcode is at the end of the sequence. If I open the fasta file and reverse complement it (barcode now at beginning of seq), the command works but then my qual file does not match (not reverse complemented). I have tried the command with a single barcode written either regularly or reverse complemented (similar to the one above) as well as with paired barcodes (barcode; seq1; seq2; sample1) but none works without reverse complementing the fasta. It seems as though I am not telling mothur to look in the right place for the barcode.

2

Why not use make.contigs on the whole dataset? Make.contigs will create a group file if you provide an oligos file. Then you could use get.groups to select the samples you want.

3

Mothur can give you the reverse of the quality file as well as a fasta file. reverse.seqs(fasta=yourFasta, qfile=yourQualityFile).