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  • tophat pair end reads

    I just finished mapping my ABI pair end colorspace reads with tophat. The reads were 50bp F3 and 25bp F5.

    When I tried to convert the .bam file into .sam for Cufflinks assembly using Picard Tools, I got this error:

    Exception in thread "main" java.lang.RuntimeException: SAM validation error: ERROR: Record 127338, Read name 507_970_1560_F3, Mate Alignment start (542169) must be <= reference sequence length (531507) on reference Contig1

    It looks like a read is being "mapped" outside of the reference contig?

    Bowtie has --fr, --rf, --ff options when aligning pair-end reads. Does tophat take into account that SOLiD mate pairs are both in forward direction? so the --ff would be used.
    Last edited by damiankao; 10-28-2010, 01:21 AM.

  • #2
    Originally posted by damiankao View Post
    I just finished mapping my ABI pair end colorspace reads with tophat. The reads were 50bp F3 and 25bp F5.

    When I tried to convert the .bam file into .sam for Cufflinks assembly using Picard Tools, I got this error:

    Exception in thread "main" java.lang.RuntimeException: SAM validation error: ERROR: Record 127338, Read name 507_970_1560_F3, Mate Alignment start (542169) must be <= reference sequence length (531507) on reference Contig1

    It looks like a read is being "mapped" outside of the reference contig?

    Bowtie has --fr, --rf, --ff options when aligning pair-end reads. Does tophat take into account that SOLiD mate pairs are both in forward direction? so the --ff would be used.

    TopHat maps left and right reads separately using Bowtie, that is, it doesn't use Bowtie's pair searching like --fr, --rf, --ff. Using the mapped reads, TopHat finds pairs if the two reads of a pair are on different strand (it ignores if they are on the same strand) and the inner distance is within user specified range.

    Comment


    • #3
      TopHat mapping outside reference contig

      Originally posted by damiankao View Post
      I just finished mapping my ABI pair end colorspace reads with tophat. The reads were 50bp F3 and 25bp F5.

      When I tried to convert the .bam file into .sam for Cufflinks assembly using Picard Tools, I got this error:

      Exception in thread "main" java.lang.RuntimeException: SAM validation error: ERROR: Record 127338, Read name 507_970_1560_F3, Mate Alignment start (542169) must be <= reference sequence length (531507) on reference Contig1

      It looks like a read is being "mapped" outside of the reference contig?

      Bowtie has --fr, --rf, --ff options when aligning pair-end reads. Does tophat take into account that SOLiD mate pairs are both in forward direction? so the --ff would be used.
      I also received this error
      Mate Alignment start must be <= reference sequence length on reference
      when trying to run picard to convert to BAM
      was this ever resolved?
      thanks,

      Comment


      • #4
        TopHat

        I was wondering why the inner distance is important in TopHat how it is related to alignment? Any one tried using variable inner distance (-r/--mate-inner-dist <int> ) and check what may be the effect on alignments, if any.

        Comment


        • #5
          I have aligned the reads using tophat version 2, but the error message remain. Why there possible exist the possition longer than the real length, in spite of the seperate alignment of tophat.

          Comment

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