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  • laxman
    Junior Member
    • Sep 2010
    • 5

    #1

    tophat 2.0.0 CIGAR and sequence length are inconsistent

    We get the following error while running Tophat 2.0.0 on a sequence. STAR aligner works fine on this sequence file. Here is the part of the log:

    [2013-04-12 10:40:14] Preparing reads
    left reads: min. length=101, max. length=101, 183210621 kept reads (230841 discarded)
    ....
    Line 171124311, sequence length 74 vs 101 from CIGAR
    Parse error at line 171124311: CIGAR and sequence length are inconsistent
    [FAILED]
    Error running bowtie:

    The offending reads looks fine to me!

    @D8CXHXP1:240:C0DBWACXX:5:1208:19908:68173 1:N:0:
    CGAGGCTGGCATTTGTGTTCTCTTAGGGTCTGCATAACATCTGTCCAGGATCTTCTGGCTTTCATATTCTCTGGTGAGAAGTCTGGTGTAATTCTAATAGG
    +
    CCCFFFFFHGHHHJJGIIGIJJJJJJIJFGGIIJJJJIJJJJJEHJJJJEIIJJJJJJJIJJJJJJJJJJJJHHH?EEFFFFFFEECECDEEFEEEEEDDD


    Any idea why this might be happening?
    we are running:
    TopHat v2.0.0
    Bowtie2 version 2.0.0-beta5
  • GenoMax
    Senior Member
    • Feb 2008
    • 7142

    #2
    You appear to be running older versions of both programs. Any possibility you can upgrade both (Tophat now in v.2.0.8 and Bowtie2 v.2.1.0) and see if the error goes away.

    Comment

    • laxman
      Junior Member
      • Sep 2010
      • 5

      #3
      i already have upgraded version but another sample from the same batch worked fine with older versions I used. I will give it a try too
      -best
      -Lax

      Comment

      • dylkot
        Junior Member
        • Jul 2013
        • 1

        #4
        I am having the exact same issue. Has anyone else seen or resolved this? In my case, the offending mapped read looks as follows:

        59 69 * 0 255 * * 0 0 CGCAAGGGCATCTCTGGGAAAGGACCTGGGGCTGGTGAGGGGCCCGGAGGAGTCTTTGCCCGCGCGTGAGACTCCA @?@BD@DDB?B;CCCF3C3AAF>AEFFB?C0?D@<)99?FFFI@;;/'=B;5(6>;>B################## ZN:Z:SRR821602.59

        and the output is:

        and the output is:

        [2013-07-18 13:34:28] Beginning TopHat run (v2.0.8b)
        -----------------------------------------------
        [2013-07-18 13:34:28] Checking for Bowtie
        Bowtie version: 2.1.0.0
        [2013-07-18 13:34:28] Checking for Samtools
        Samtools version: 0.1.19.0
        [2013-07-18 13:34:28] Checking for Bowtie index files
        [2013-07-18 13:34:28] Checking for Bowtie index files
        [2013-07-18 13:34:28] Checking for reference FASTA file
        [2013-07-18 13:34:28] Generating SAM header for /home/unix/dylkot/genomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome
        format: fastq
        quality scale: phred33 (default)
        [2013-07-18 13:35:31] Reading known junctions from GTF file
        [2013-07-18 13:35:37] Preparing reads
        left reads: min. length=76, max. length=76, 2500 kept reads (0 discarded)
        right reads: min. length=76, max. length=76, 2498 kept reads (2 discarded)
        [2013-07-18 13:35:37] Using pre-built transcriptome index..
        [2013-07-18 13:35:41] Mapping left_kept_reads to transcriptome genome with Bowtie2
        [2013-07-18 13:35:49] Mapping right_kept_reads to transcriptome genome with Bowtie2
        [2013-07-18 13:35:56] Reporting output tracks
        [FAILED]
        Error running /home/unix/dylkot/bin/tophat-2.0.8b.Linux_x86_64/tophat_reports --min-anchor 8 --splice-mismatches 0 --min-report-intron 50 --max-report-intron 500000 --min-isoform-fraction 0.15 --output-dir /broad/hptmp/dkotliar/GTEX/mapped/debug/ --max-multihits 20 --max-seg-multihits 40 --segment-length 25 --segment-mismatches 2 --min-closure-exon 100 --min-closure-intron 50 --max-closure-intron 5000 --min-coverage-intron 50 --max-coverage-intron 20000 --min-segment-intron 50 --max-segment-intron 500000 --read-mismatches 5 --read-gap-length 2 --read-edit-dist 7 --read-realign-edit-dist 8 --max-insertion-length 3 --max-deletion-length 3 -z gzip --inner-dist-mean 67 --inner-dist-std-dev 231 --gtf-annotations /home/unix/dylkot/genomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome.gff --gtf-juncs /home/unix/dylkot/mapped/debug/tmp/genome.juncs --no-closure-search --no-coverage-search --no-microexon-search --sam-header /home/unix/dylkot/mapped/debug/tmp/genome_genome.bwt.samheader.sam --report-discordant-pair-alignments --report-mixed-alignments --samtools=/home/unix/dylkot/bin/samtools-0.1.19/samtools --bowtie2-max-penalty 6 --bowtie2-min-penalty 2 --bowtie2-penalty-for-N 1 --bowtie2-read-gap-open 5 --bowtie2-read-gap-cont 3 --bowtie2-ref-gap-open 5 --bowtie2-ref-gap-cont 3 /home/unix/dylkot/genomes/Homo_sapiens/UCSC/hg19/Sequence/Bowtie2Index/genome.fa /home/unix/dylkot/mapped/debug/junctions.bed /home/unix/dylkot/mapped/debug/insertions.bed /home/unix/dylkot/mapped/debug/deletions.bed /home/unix/dylkot/mapped/debug/fusions.out /home/unix/dylkot/mapped/debug/tmp/accepted_hits /home/unix/dylkot/mapped/debug/tmp/left_kept_reads.m2g.bam /home/unix/dylkot/mapped/debug/tmp/left_kept_reads.bam /home/unix/dylkot/mapped/debug/tmp/right_kept_reads.m2g.bam //home/unix/dylkot/mapped/debug/tmp/right_kept_reads.bam
        Error: CIGAR and sequence length are inconsistent!(CGCAAGGGCATCTCTGGGAAAGGACCTGGGGCTGGTGAGGGGCCCGGAGGAGTCTTTGCCCGCGCGTGAGACTCCA)

        Any idea what could be going on or how I can best get around this problem?

        Comment

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