I use the 2.0 version of mothur to deal with about 50 of sanger sequencing sequences (from a clone library to check if we mixed the DNA from different samples well enough) which have barcode within them. when i use the trim.seqs command, mothur says “Error opening file: Invalid argument 49”, but it output three files(trim.fasta, scrap.fasta and groups), and all the sequences are winded up in the scrap file, the reason is mothur can’t find the barcode (|bfr, you know it). I doubt if mothur can’t read the oligos file ? when i add a # before the forward and reverse primer (to tell mothur to ignore these two lines), trim.seqs again, and the scrap file says can’t find the barcode (|b, you know it), so i think mothur can read the oligos file. So i have no idea, what’s wrong. Help and thanks!
forward TCGGACTAC
reverse CAGTGCCAGC
barcode AACGAACG 1-1
barcode AACGAAGC 1-2
barcode AACGATCC 1-3
barcode AACGATGG 1-4
barcode AACGCCAT 1-5
barcode AACGCCTA 3-1
barcode AACGCGAA 3-2
barcode AACGCGTT 3-3
barcode AACGGCAA 3-4
barcode AACGGCTT 3-5
barcode AACGTACC 5-1
barcode AACGTAGG 5-2
barcode AACGTTCG 5-3
barcode AACGTTGC 5-4
barcode AAGCAACG 5-5
barcode AAGCAAGC 7-1
barcode AAGCATCC 7-2
barcode AAGCATGG 7-3
barcode AAGCCGAA 7-4
barcode AAGCCGTT 7-5
barcode AAGCGCAA 8-1
barcode AAGCGCTT 8-2
barcode AAGCGGAT 8-3
barcode AAGCGGTA 8-4
barcode AAGCTACC 8-5
barcode AAGCTAGG 9-1
barcode AAGCTTCG 9-2
barcode AAGCTTGC 9-3
barcode AAGGAACC 9-4
barcode AAGGAAGG 9-5
barcode AAGGATCG 10-1
barcode AAGGATGC 10-2
barcode AAGGCCAA 10-3
barcode AAGGCCTT 10-4
barcode AAGGCGAT 10-5