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