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  • tophat rna-seq crash

    I'm using tophat for aligning Illumina rna-seq data. It crashed in one of the last steps:

    [Tue Jan 10 08:26:56 2012] Beginning TopHat run (v1.4.0)
    -----------------------------------------------
    [Tue Jan 10 08:26:56 2012] Preparing output location ./tophat_out_bwa/
    [Tue Jan 10 08:26:56 2012] Checking for Bowtie index files
    [Tue Jan 10 08:26:56 2012] Checking for reference FASTA file
    [Tue Jan 10 08:26:56 2012] Checking for Bowtie
    Bowtie version: 0.12.7.0
    [Tue Jan 10 08:26:56 2012] Checking for Samtools
    Samtools Version: 0.1.17
    [Tue Jan 10 08:26:56 2012] Generating SAM header for hshiv
    format: fastq
    quality scale: phred33 (default)
    [Tue Jan 10 08:27:16 2012] Preparing reads
    left reads: min. length=101, count=86332313
    [Tue Jan 10 08:55:48 2012] Mapping left_kept_reads against hshiv with Bowtie
    [Tue Jan 10 09:35:06 2012] Processing bowtie hits
    [Tue Jan 10 10:30:04 2012] Mapping left_kept_reads_seg1 against hshiv with Bowtie (1/4)
    [Tue Jan 10 12:41:18 2012] Mapping left_kept_reads_seg2 against hshiv with Bowtie (2/4)
    [Tue Jan 10 14:54:35 2012] Mapping left_kept_reads_seg3 against hshiv with Bowtie (3/4)
    [Tue Jan 10 17:08:07 2012] Mapping left_kept_reads_seg4 against hshiv with Bowtie (4/4)
    [Tue Jan 10 18:56:53 2012] Searching for junctions via segment mapping
    [FAILED]
    Error: segment-based junction search failed with err =1
    Error: could not get read# 839911731 from stream!

    I used tophat on Ubuntu Linux (linux kernel 3.0.0-14-generic). Actually, I first aligned my reads with bwa (bwa aln, then bwa samse) and extracted the unmapped reads with samtools into bam. From there, I used the (tophat) bam2fastx tool to convert the unmapped reads back into fastq. The unmapped reads (about 30%) then go as Input into tophat.

    Has someone any idea what's going on there?

    Thanks for your help
    Wolfgang

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