Java error running rnaseq modeule with qualimap 2.0

[Tom Harrop]

Hi,

I've just installed Qualimap 2.0 on a headless cluster. I'm trying to run the rnaseq module but it's failing with the following java error:

Initializing regions from sp/genome/spiro_v2_cuffcomp.gtf...

Initialized 100000 regions...
Failed to run rnaseq

java.lang.IllegalArgumentException: start must be less than or equal to end!
    at net.sf.picard.util.Interval.<init>(Interval.java:74)
    at net.sf.picard.util.Interval.<init>(Interval.java:52)
    at org.bioinfo.ngs.qc.qualimap.common.TranscriptDataHandler.createHelperMaps(TranscriptDataHandler.java:674)
    at org.bioinfo.ngs.qc.qualimap.common.TranscriptDataHandler.constructTranscriptsMap(TranscriptDataHandler.java:174)
    at org.bioinfo.ngs.qc.qualimap.process.ComputeCountsTask.loadRegionsFromGTF(ComputeCountsTask.java:565)
    at org.bioinfo.ngs.qc.qualimap.process.ComputeCountsTask.initRegions(ComputeCountsTask.java:474)
    at org.bioinfo.ngs.qc.qualimap.process.ComputeCountsTask.run(ComputeCountsTask.java:383)
    at org.bioinfo.ngs.qc.qualimap.process.RNASeqQCAnalysis.run(RNASeqQCAnalysis.java:67)
    at org.bioinfo.ngs.qc.qualimap.main.RnaSeqQcTool.execute(RnaSeqQcTool.java:135)
    at org.bioinfo.ngs.qc.qualimap.main.NgsSmartTool.run(NgsSmartTool.java:187)
    at org.bioinfo.ngs.qc.qualimap.main.NgsSmartMain.main(NgsSmartMain.java:103)

I've tried three different GTF files and I'm getting the error with all of them. I've previously run older versions of Qualimap using the same GTF files without any problems.

Here's an example command:

qualimap rnaseq -bam cr/tophat/295.1.trimmed.tophat/accepted_hits.bam -gtf cr/genome/Creinhardtii_236_gene_exons_cuffcomp.gtf -outdir cr/qualimap/raw -outfile cr/qualimap/295.1.rnaseqc.html -p non-strand-specific

Please let me know if you need any more information.

Thanks,

Tom

[Konstantin Okonechnikov]

Hi Tom,

thanks for your report!

Could you please share a problematic GTF file or at least a subset from it on which the issue is reproducible? That would be the easiest way to look into it.

Best regards,

   Konstantin

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[Tom Harrop]

Hi Konstantin,

Thanks for the reply. I'm sending you a link to a GTF that I uploaded to dropbox. I'll use direct message, so if you don't get the link it means it got eaten by the Google Groups monster, just let me know and I'll try again.

Tom

[Konstantin Okonechnikov]

Tom,

thanks a lot, I got the data, will look into into it.

--

  Konstantin

--

[Konstantin Okonechnikov]

Hi Tom,

the problem was with the order of exons for transcripts from reverse strand.

I fixed the issue and uploaded the snapshot:
https://www.dropbox.com/s/95608xsa5j4iq83/qualimap-build-19-09-14.tar.gz?dl=0

Could you please test it and see if it works now with your data now?

[twha...@gmail.com]

Hi Konstantin,

Looks like it's fixed. It ran fine with the exact command I pasted into the first post. Thanks a lot!

Tom

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[roberto....@gmail.com]

Hi Konstantin,

I am having Tom's same issue. I initially used Qualimap 2.0 and now Qualimap 2.0.1, both complain about start that must be equal or less than end. However:

roberto@seqC:~$ awk '$4==$5 {print $0}' sorted.gtf | wc -l

175

roberto@seqC:~$ awk '$4<$5 {print $0}' sorted.gtf | wc -l

664339

roberto@seqC:~$ awk '$4>$5 {print $0}' sorted.gtf | wc -l

0

roberto@seqC:~$ cat sorted.gtf | wc -l

664514

This is my GTF file:

https://dl.dropboxusercontent.com/u/8992918/sorted.gtf.zip

Thank you for your help,

Roberto

[Konstantin Okonechnikov]

Hi Roberto!

Thanks for the report!

The problem was because the GTF file inlcudes some genes from different chromosomes that have the same transcript_Id

For example check this one:

$ grep "transcript_id \"NM_011335\"" sorted.gtf | grep exon

chr4    mm10_refGene    exon    41903649    41903751    0.000000    +    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4    mm10_refGene    exon    41903840    41903960    0.000000    +    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4    mm10_refGene    exon    41904055    41904237    0.000000    +    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4    mm10_refGene    exon    41904320    41904743    0.000000    +    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_GL456350_random    mm10_refGene    exon    1    424    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_GL456350_random    mm10_refGene    exon    507    689    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_GL456350_random    mm10_refGene    exon    784    904    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_GL456350_random    mm10_refGene    exon    993    1095    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_JH584293_random    mm10_refGene    exon    59769    60192    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_JH584293_random    mm10_refGene    exon    60275    60457    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_JH584293_random    mm10_refGene    exon    60552    60672    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";
chr4_JH584293_random    mm10_refGene    exon    60761    60863    0.000000    -    .    gene_id "NM_011335"; transcript_id "NM_011335";

I will try to fix this problem as soon as possible.

Meanwhile try using this version:
https://www.dropbox.com/s/azray3duqh82gft/qualimap-build-11-01-15.tar.gz

Here such repeating transcripts are skipped.

--
  Konstantin

[roberto....@gmail.com]

Thanks Konstantin,

I will use the dev version meanwhile.

Roberto

[Morgane Roth]

Thank you very much Konstantin !
I had a similar bug and it solved it.
Cheers
Morgane

[Roy Francis]

I got this error as below and I was wondering if this might be related to the gtf file as well.

Thanks,

Roy

Java memory size is set to 1200M
Launching application...


Java HotSpot(TM) 64-Bit Server VM warning: ignoring option MaxPermSize=1024m; support was removed in 8.0
QualiMap v.2.0.2
Built on 2015-02-10 11:39


Selected tool: rnaseq
Initializing regions from /home/royfranc/ngs/common-data/genomic-data-ensembl/gene-set-gtf/Danio_rerio.GRCz10.83.gtf...


Failed to run rnaseq


java.lang.NullPointerException
        at org.bioinfo.ngs.qc.qualimap.common.GenomicFeatureStreamReader.readNextRecord(GenomicFeatureStreamReader.java:188)
        at org.bioinfo.ngs.qc.qualimap.process.ComputeCountsTask.loadRegionsFromGTF(ComputeCountsTask.java:544)
        at org.bioinfo.ngs.qc.qualimap.process.ComputeCountsTask.initRegions(ComputeCountsTask.java:478)
        at org.bioinfo.ngs.qc.qualimap.process.ComputeCountsTask.run(ComputeCountsTask.java:387)
        at org.bioinfo.ngs.qc.qualimap.process.RNASeqQCAnalysis.run(RNASeqQCAnalysis.java:67)
        at org.bioinfo.ngs.qc.qualimap.main.RnaSeqQcTool.execute(RnaSeqQcTool.java:163)