invalid DNA sequence in mapping file, degenerate primers

[Theresa]

Hi Tony,

   I was trying to extract barcodes and got the following error:

    extract_barcodes.py -f /home/qiime/Desktop/COI_modified/raw\ sequence\ files/SAM1-3_S8_L001_R1_001.fastq -r /home/qiime/Desktop/COI_modified/raw\ sequence\ files/SAM1-3_S8_L001_R2_001.fastq -c barcode_paired_end -m /home/qiime/Desktop/COI_modified/validate_mapping_file_output/modified_COI_with_Reverse_primer_corrected.txt --attempt_read_reorientation --bc1_len 8 --bc2_len 0 -o /home/qiime/Desktop/COI_modified/processed_seqs_removedbarcodesTraceback (most recent call last):

  File "/usr/local/bin/extract_barcodes.py", line 175, in <module>

    main()

  File "/usr/local/bin/extract_barcodes.py", line 171, in main

    opts.attempt_read_reorientation, disable_header_match)

  File "/usr/local/lib/python2.7/dist-packages/qiime/extract_barcodes.py", line 79, in extract_barcodes

    forward_primers, reverse_primers = get_primers(header, mapping_data)

  File "/usr/local/lib/python2.7/dist-packages/qiime/extract_barcodes.py", line 540, in get_primers

    primer in raw_reverse_primers])

  File "/usr/local/lib/python2.7/dist-packages/skbio/sequence/_sequence.py", line 1698, in reverse_complement

    return self._complement(reverse=True)

  File "/usr/local/lib/python2.7/dist-packages/skbio/sequence/_sequence.py", line 1612, in _complement

    "%s.complement_map?" % (base, self.__class__.__name__))

skbio.sequence._exception.BiologicalSequenceError: Don't know how to complement base I. Is it in DNASequence.complement_map?

It turns out that my mapping file has an "invalid DNA sequence" (attached).  I am not sure what to do because it is a degenerate primer as is the forward primer which wasn't picked up as an invalid sequence.  The problem is that I need the reverse primer in there because I use extract barcodes twice, once to extract the barcodes and then the primers (after joining pairs) before I attempt to split the libraries.  Is there a work around for degenerate primers that come up as an invalid DNA sequence?

Thanks!

Theresa

[TonyWalters]

Hello,

Inosine isn't coded as a nucleotide. I think you'd want to go with the degenerate base D (which is equivalent to A/G/T) in this case, which should cover the regular binding of inosine to other nucleotides.