Question: 454 Sequences: Lower Case Base Call In Reverse Primer
3
gravatar for lgbi
5.3 years ago by
lgbi150
lgbi150 wrote:

After converting a 454 SFF file to FASTQ, I get sequences like this:

tcagAGTACGCTATGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCATTATAACCACTCAAGCTCTCGCTTGGTATTGGGGCTCGCGGTTTCGCGGCTCCTAAAATCAGTGGCGGTGCCTATCGGCTCTACGCGTAGTAATACTCCTCGCGATTGAGTCCGGTAGGTCTACTTGCCAGCAACCCCTAATTTTTTTAAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGactgagactgccaaggcacacaggggataggnn

Key sequence, barcode, forward primer are all OK, but the last nucleotide of (the reverse complement of) the reverse primer is always lower case: GCATATCAATAAGCGGAGGa.

Because of this, it gets deleted when I do a trimmed conversion from SFF to FASTQ, and the sequence gets lost when my software looks for primers and barcodes. This has been happening for several 454 runs the last couple of months.

I can of course easily work around this, but still I would like to know what is causing this. Anybody has an idea?

454 • 1.8k views
ADD COMMENTlink modified 2.6 years ago by Biostar ♦♦ 20 • written 5.3 years ago by lgbi150

what tool do you use to convert the SFF to FASTA?

ADD REPLYlink written 5.3 years ago by Istvan Albert ♦♦ 78k

Very good question - if the conversion tool had an off by one error, that would explain the 'extra' base being clipped.

ADD REPLYlink written 5.3 years ago by Peter5.7k

I'm using the sffinfo tool provided by Roche.

ADD REPLYlink written 5.3 years ago by lgbi150

Have you asked your sequence provider if they know about this issue?

ADD REPLYlink written 5.3 years ago by Peter5.7k
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