Hello Lindsey,

Would you please send in a bug report from the FastQC error dataset that resulted from the run where you included running the Groomer tool first? This will help us to track down the problem.

Please be sure to leave all datasets in your history for this run undeleted and in the original history that they were executed in. If you need to undelete, use "Option (gear icon) -> View Deleted Datasets", the click on the links to undelete. We will need the original inputs and full path through tools run in Galaxy.
http://wiki.g2.bx.psu.edu/Support#Reporting_tool_errors

Please include this email address in the bug report notes if your Galaxy account uses a different one.

Thanks and I will watch for your bug report,

Jen
Galaxy team

On 7/11/12 5:32 AM, Lindsey Kelly wrote:
Good Morning,

I confirmed with Illumina that my reads are in Sanger FASTQ format and
used edit attributes to change them to fastqsanger.  Then I joined the
forward and reverse and ran the FastQ Joiner and the FastQC on the
joined data file.  I'm getting the following error:

## odpath=None: No output found in None. Output for the run was:

Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/space/g2main
Started analysis of FASTQ
Analysis complete for FASTQ
Failed to process file FASTQ
java.lang.IllegalArgumentException: No known encodings with chars < 33 (Yours 
was )
        at 
uk.ac.bbsrc.babraham.FastQC.Sequence.QualityEncoding.PhredEncoding.getFastQEncodingOffset(PhredEncoding.java:33)
        at 
uk.ac.bbsrc.babraham.FastQC.Modules.PerBaseQualityScores.getPercentages(PerBaseQualityScores.java:70)
        at 
uk.ac.bbsrc.babraham.FastQC.Modules.PerBaseQualityScores.raisesError(PerBaseQualityScores.java:164)
        at 
uk.ac.bbsrc.babraham.FastQC.Report.HTMLReportArchive.startDocument(HTMLReportArchive.java:194)
        at 
uk.ac.bbsrc.babraham.FastQC.Report.HTMLReportArchive.(HTMLReportArchive.java:59)
        at 
uk.ac.bbsrc.babraham.FastQC.Analysis.OfflineRunner.analysisComplete(OfflineRunner.java:157)
        at 
uk.ac.bbsrc.babraham.FastQC.Analysis.AnalysisRunner.run(AnalysisRunner.java:108)
        at java.lang.Thread.run(Thread.java:662)

I thought that perhaps this was because I just changed the file format and 
maybe they really aren't in Fastqsanger format, so I: changed the format back 
to fastq, ran the groomer, joined them and then ran the FastQC and received the 
same error.

I also tried to join them first and then run the groomer, but the files don't 
show up if I haven't changed the attributes to Fastqsanger (which makes sense 
if Galaxy only uses fastqsanger files).

I'm not sure what to try next.

Thank you
Lindsey


On Wed, Jul 4, 2012 at 3:53 PM, Jennifer Jackson <j...@bx.psu.edu
<mailto:j...@bx.psu.edu>> wrote:

    Hello Lindsey,

    Yes, you have this correct. The general path would be to:

      - join forward and reverse data per run
      - run FASTQ Groomer & FastQC
        (note: if your data is already in Sanger FASTQ format with
    Phred+33 quality scaled
        values, the datatype '.fastqsanger' can be directly assigned and
    the FASTQ Groomer
       step skipped. This is likely true if your data is a from the
    latest CASAVA pipeline, but
        please double check.)
      - discard data as needed based on quality
      - split forward and reverse data that passes QC
      - concatenate all forward reads from a sample into one FASTQ file
      - concatenate all reverse reads from a sample into one FASTQ file.
      - for each sample, run TopHat using the two concatenated FASTQ files

    To manipulate paired end data, please see the tools -> NGS: QC and
    manipulation: FASTQ splitter & FASTQ joiner.

    To combined data files head-to-tail from multiple runs into a single
    FASTQ file please see the tool -> Text Manipulation: Concatenate
    datasets.

    I am not sure of the actual volume of data, but if these start to
    get large or TopHat errors with a memory problem, a local or cluster
    instance would be the recommendation: http://getgalaxy.org

    For reference:
    http://tophat.cbcb.umd.edu/manual.html
    http://www.nature.com/nprot/journal/v7/n3/full/nprot.2012.016.html

    Hopefully this helps. Others are welcome to post comments/suggestions.

    Jen
    Galaxy team


    On 7/2/12 11:17 AM, Lindsey Kelly wrote:

    I am trying to do RNAseq analysis on Paired end data from the
    Hiseq2000.  I have about 50 files for each sample (25 forward and
    25 reverse - although each sample has a different number of files).

    I think that I need to:

    -convert them into FASTQ sanger format using the FASTSQ groomer tool

    -check the quality using the FASTQqc tool

    I don't know how to handle this many files. Do I have to groom and
    run the QC for each file? Should I join the paired files and run
    both tools on each pair, or should I combine all of the data for
    each sample (which I don't know how to do) and then groom and run
    the QC for all of the reads for the sample.


    Thanks in advance for advice

    Lindsey



    ___________________________________________________________
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    http://galaxyproject.org






--

Lindsey M Kelly
University of Pittsburgh
Cellular and Molecular Pathology Program
Yuri Nikiforov Laboratory
412-578-9208


--
Jennifer Jackson
http://galaxyproject.org


___________________________________________________________
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