That's a neat trick, and I definitely wouldn't have thought of that
approach, so thanks for that!

After I finished writing this out, I realized it was super long.  So here
are the questions I'm asking up front, so you can choose whether or not to
read the details.  Thanks!

1. How do I output the quality scores when converting from FASTQ to FASTA?
2. Does the SAM-to-interval tool output only mapped reads by looking at the
flag values?
3. Why am I getting the mentioned error and is there a way to resolve it?

Here are the details:

   1. I don't see an option to output both the sequences and the quality
   scores.  I found two FASTQ-to-FASTA converters (one under the "Convert
   Formats" and the other in the FastX Toolkit) and both only output one fasta
   file with the sequences.  Am I missing something, or should I be using some
   other tool to output both the sequences and the quality scores?
   2. The Extract Genomic Sequences tool seems to want an Interval file as
   input, not a list of IDs.  Does that mean I should convert the filtered SAM
   output to Interval?  Currently I'm using the SAM-to-interval conversion to
   extract the mapped reads and make the data more manageable in one step
   (pretty sure I picked that up from one of the tutorials...).  I was
   assuming that by definition it could only output an interval if it was
   mapped, and if so, I wouldn't be able to convert the unmapped reads to
   Interval anyway.  Is that wrong?
   3. I was setting up a workflow with Bowtie and I noticed that the
   Workflow Editor does show options to output unmapped reads.  But when I try
   to output them, I get this error:

"Error due to input mapping of 'Compute quality statistics' in
'output_unmapped_reads_l'. A common cause of this is conditional outputs
that cannot be determined until runtime, please review your workflow."

Superficially, this seems silly.  Obviously a "conditional output" will not
be determined until runtime because it's dependent on something else.  So
why is that an error?  I have tried outputting to a few different tools, so
it doesn't seem to be specific to the tool into which the unmapped reads go
(in this case, "Compute Quality Statistics").


Any thoughts, insights, or even other approaches to the original problem
would be great.  Currently, I'm thinking my best bet is to filter out the
unmapped reads locally with a Perl script and re-upload, but that felt like
overkill and time-consuming when I will inevitably want to tweak or re-run
things.  Also, installing a local instance is currently not an option for
me (though it should be in a few months). In any case, I appreciate your
help a lot!

Thanks, again!
Mayank Tandon


On Thu, Jul 18, 2013 at 5:38 PM, Jennifer Jackson <j...@bx.psu.edu> wrote:

>  Hi Mayank,
>
> The best option I know of is to do the following:
>
> 1 - obtain the sequence identifiers for the unmapped reads by filter the
> SAM file, then cutting them out
> 2 - convert the original FASTQ file to FASTA - you should get two output,
> one for the sequences and one for the quality score values
> 3 - use the tool "Fetch Sequences -> Extract Genomic DNA". The query is
> the list from #1, the target is the "genome" from #2. Do this twice - once
> for seqs, once for quals. This means using the target datasets from #2 as
> Custom Reference genomes - help about how to do this is here:
> http://wiki.galaxyproject.org/Support#Custom_reference_genome
> 4 - combine the FASTA seq and qual files back to FASTQ
>
> If you will be doing this again, then capture the process into a workflow
> for future use, in a way creating your own "tool".
>
> Hopefully this helps!
>
> Jen
> Galaxy
>
>
> On 7/18/13 9:40 AM, Mayank Tandon wrote:
>
> I was hoping this question had been asked, but I haven't been able to find
> it.  I want to output the unmapped reads from bowtie as a fastq file for
> subsequent mapping to other genomes (i.e. the "--un <filename>" option).  I
> know I can extract the unmapped reads by filtering on the bitwise values in
> the sam output and converting to fastq with the Picard tool, but I'm using
> colorspace data and bowtie converts them to letterspace. My understanding
> (coming mostly from forums and personal discussions) was that the
> color-to-letter conversion was somehow lossy so mapping the colorspace data
> directly is always preferable.
>
>  So the question is: Is bowtie's '--un' option implemented in Galaxy and
> if so, how do I access it?
>
>  Thanks in advance!
>
>
>  Mayank Tandon
>
>
> ___________________________________________________________
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> --
> Jennifer Hillman-Jackson
> Galaxy Support and Traininghttp://galaxyproject.org
>
>
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