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  • adisaxena
    Junior Member
    • Aug 2015
    • 6
    #1

    TopHat2 Help-Error: segment-based junction search failed with err =-11

    Hi all,

    I am new to Tuxedo tools and want to use TopHat + Cufflinks for differential gene expression analysis with RNASeq data.

    I have ~77.7 million mouse SR-50 illumina reads (libraries prepared with strand-specific TrueSeq mRNA, illumina kit) that I want to map back to the Mouse genome. I use prebuild iGenome mouse index (NCBI_Build37.2) as a reference and have tried to run the Tophat pipeline on a cluster (two attempt with memory=120Gb;CPU=24 and memory=700Gb;CPU-30) and got this error:

    [2016-05-29 13:08:56] Beginning TopHat run (v2.1.0)
    -----------------------------------------------

    [2016-05-29 13:08:56] Checking for Bowtie Bowtie version: 2.2.4.0

    [2016-05-29 13:08:56] Checking for Bowtie index files (genome)..

    [2016-05-29 13:08:56] Checking for reference FASTA file

    [2016-05-29 13:08:56] Generating SAM header for /oasis/projects/nsf/csd473/adi/Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome

    [2016-05-29 13:09:05] Reading known junctions from GTF file

    [2016-05-29 13:09:07] Preparing reads

    left reads: min. length=40, max. length=50, 77774302 kept reads (3761 discarded)

    [2016-05-29 13:18:23] Building transcriptome data files M1M_TopHat2_MusGenBuild37.2/tmp/genes

    [2016-05-29 13:18:47] Building Bowtie index from genes.fa

    [2016-05-29 13:21:03] Mapping left_kept_reads to transcriptome genes with Bowtie2

    [2016-05-29 13:39:09] Resuming TopHat pipeline with unmapped reads

    [2016-05-29 13:39:09] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2

    [2016-05-29 13:41:31] Mapping left_kept_reads.m2g_um_seg1 to genome genome with Bowtie2 (1/2)

    [2016-05-29 13:42:03] Mapping left_kept_reads.m2g_um_seg2 to genome genome with Bowtie2 (2/2)

    [2016-05-29 13:42:34] Searching for junctions via segment mapping

    [FAILED]

    Error: segment-based junction search failed with err =-11

    My run script is:-

    tophat2 -p 30 -G Build37.2_iGenome_Mus/Annotation/Archives/archive-2015-07-17-14-32-40/Genes/genes.gtf --library-type fr-secondstrand --no-coverage-search -o TopHat2_MusGenBuild37.2 Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome M1M_Trimmed.fastq.gz

    Could someone please help me understand the error 'segment-based junction search failed with err =-11'? The 'segment_juncs.log' file is blank.

    Shall be very grateful for your help with troubleshoot advice!

    thanks in advance,
    Adi
    Tags: None
    • pepperoni
      Member
      • Oct 2011
      • 59
      #2
      Originally posted by adisaxena View Post
      Hi all,

      I am new to Tuxedo tools and want to use TopHat + Cufflinks for differential gene expression analysis with RNASeq data.

      I have ~77.7 million mouse SR-50 illumina reads (libraries prepared with strand-specific TrueSeq mRNA, illumina kit) that I want to map back to the Mouse genome. I use prebuild iGenome mouse index (NCBI_Build37.2) as a reference and have tried to run the Tophat pipeline on a cluster (two attempt with memory=120Gb;CPU=24 and memory=700Gb;CPU-30) and got this error:

      [2016-05-29 13:08:56] Beginning TopHat run (v2.1.0)
      -----------------------------------------------

      [2016-05-29 13:08:56] Checking for Bowtie Bowtie version: 2.2.4.0

      [2016-05-29 13:08:56] Checking for Bowtie index files (genome)..

      [2016-05-29 13:08:56] Checking for reference FASTA file

      [2016-05-29 13:08:56] Generating SAM header for /oasis/projects/nsf/csd473/adi/Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome

      [2016-05-29 13:09:05] Reading known junctions from GTF file

      [2016-05-29 13:09:07] Preparing reads

      left reads: min. length=40, max. length=50, 77774302 kept reads (3761 discarded)

      [2016-05-29 13:18:23] Building transcriptome data files M1M_TopHat2_MusGenBuild37.2/tmp/genes

      [2016-05-29 13:18:47] Building Bowtie index from genes.fa

      [2016-05-29 13:21:03] Mapping left_kept_reads to transcriptome genes with Bowtie2

      [2016-05-29 13:39:09] Resuming TopHat pipeline with unmapped reads

      [2016-05-29 13:39:09] Mapping left_kept_reads.m2g_um to genome genome with Bowtie2

      [2016-05-29 13:41:31] Mapping left_kept_reads.m2g_um_seg1 to genome genome with Bowtie2 (1/2)

      [2016-05-29 13:42:03] Mapping left_kept_reads.m2g_um_seg2 to genome genome with Bowtie2 (2/2)

      [2016-05-29 13:42:34] Searching for junctions via segment mapping

      [FAILED]

      Error: segment-based junction search failed with err =-11

      My run script is:-

      tophat2 -p 30 -G Build37.2_iGenome_Mus/Annotation/Archives/archive-2015-07-17-14-32-40/Genes/genes.gtf --library-type fr-secondstrand --no-coverage-search -o TopHat2_MusGenBuild37.2 Build37.2_iGenome_Mus/Sequence/Bowtie2Index/genome M1M_Trimmed.fastq.gz

      Could someone please help me understand the error 'segment-based junction search failed with err =-11'? The 'segment_juncs.log' file is blank.

      Shall be very grateful for your help with troubleshoot advice!

      thanks in advance,
      Adi
      Hi, did you solved this?
      Any advice will be appreciated

        Comment

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