Myseq - demultiplexing paired barcodes and primers

Commands in mothur
1

Hi,
I have Myseq paired end reads with barcodes at both ends (after merging fwd and rev) + primers, and I want to demultiplex at sample/barcode level trimming the barcodes and primers.

When I do the commands: trim.seqs and fastq.info it gives me an error: “cannot mix paired primers and barcodes with non paired or linkers and spacers, quitting.”

the oligos file looks like this:

forward GTGARTCATCGARTCTTTG
reverse TCCTCCGCTTATTGATATGC
barcode NAACAAC NNAACAAC x1
barcode NNAACCGA NNNAACCGA x2

the command looks like this:

trim.seqs(fasta=my_Assembled_Read.fasta, oligos=My_Oligos_File.oligos, checkorient=T)

I don’t understand if the configuration of my oligos file is wrong or if it’s not possible to trim in one go paired barcodes and primers…
Thank you in advance for your help.
Cheers

2

You want to use the ‘primer’ tag to indicate to mothur you have both forward and reverse primers.

primer GTGARTCATCGARTCTTTG TCCTCCGCTTATTGATATGC
barcode NAACAAC NNAACAAC x1
barcode NNAACCGA NNNAACCGA x2