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> 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
>
>
> ___________________________________________________________
> The Galaxy User list should be used for the discussion of
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>
>
> --
> Jennifer Jacksonhttp://galaxyproject.org
>
>
>
>


-- 

Lindsey M Kelly
University of Pittsburgh
Cellular and Molecular Pathology Program
Yuri Nikiforov Laboratory
412-578-9208
___________________________________________________________
The Galaxy User list should be used for the discussion of
Galaxy analysis and other features on the public server
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