Hi Daniel,

Thanks for reporting this issue - we have done some testing here and can duplicate your results.

We are still reviewing options about how to address this, including Galaxy wrapper modifications and possibly more. The issue has to do with how certain variables are passed in the wrapper and interpreted in the command string. If you are interested, write me back to let me know and I can send you a link to the development ticket you can track (once created).

Back to how to do this sort of analysis - yes, "aln -n" is a type of mismatch parameter. And ideally setting this would result in a mismatch-free alignment. Instead it returns no results:

Maximum edit distance (aln -n):  "0" 
and
Fraction of missing alignments given 2% uniform base error rate (aln -n): "0"

As a work-around, we have found that making the the Fraction variable very small achieves a close approximation of mismatch-free alignments on our test sets. This is by no means a guarantee, but pending future changes, these are the recommended form settings:

Maximum edit distance (aln -n):  "0" 
and
Fraction of missing alignments given 2% uniform base error rate (aln -n): "0.00001"

Thank you for your patience while we worked out exactly what was going on. Hopefully the temporary work-around will allow you to continue with your research,

Jen
Galaxy team

On 3/2/13 11:44 AM, Daniel Sher wrote:

Hello,
We have a sample containing several bacterial species and we want to uniquely map RNA-seq reads to the genomes of each of our organisms to get the expression patterns of each organism separately. We tried to use BWA in Galaxy with the “edit distance” (aln -n in the command line version) set to 0 but none of the reads were mapped (all had the SAM tag set to “4’). This is an artifact since running BLAST with some of the sequences showed that they have 100% identity to one of our genomes and not any others, so they should map uniquely.

When running BWA with the number of mismatches set to between 1-5 >90% of our reads were mapped, and the number of mapped reads increased with the mismatch number so that seems to be working OK.

Does the "aln -n" option really determine the number of mismatches? Any ideas why BWA will not run well in Galaxy using –n=0?
Thanks
Daniel

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