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  • MRNM problem for the .sam output file of tophat

    Hi everyone,
    MRNM is the 7th field in the sam file. I got the sam output from tophat, it seems that the MRNM is not correct in my case.

    $ grep "HWI-EAS244_1_1_116_848_845_0_1_12496949" accepted_hits.sam
    HWI-EAS244_1_1_116_848_845_0_1_12496949;2 163 chr1 14628 255 75M = 14859 0 CCTGGCTGTGTCCATGTCAGAGCAACGGCCCAAGTCTGGGTCTGGGGGGGCAGGTGTCATGGAGCCCCCTACGCT ABA?BBBB=A>BABA@2@B=;;=9<AB8ABB38=0>A?<=4AA<9??=########################### NM:i:2
    HWI-EAS244_1_1_116_848_845_0_1_12496949;1 83 chr9 14859 255 76M = 14628 0 GGCAAAGGAGGGATGGAGTCTGACACGCGGACAAAGGCTCCTCCGGGCCCCTCACCAGCCCCAGGTCCTTTCCCAG 7>@@)@B=1@B;<(8@?@@9@A>B@BBABAAA@AAAB@@A@<AABABBBA?BB@BBBBB@BABCBCCCCCCCCCCB NM:i:0


    The MRNM is '=' here, but one is on chr1 and another is on chr9.

    From the manual , if MRNM is '=', the mate pairs should be on the same reference(same chromosome), but here it's on different chromosome. Does anybody meet the same problem? How to solve this problem?

    version of tophat: tophat1.0.13
    part of the shell code for tophat running is:
    "
    tophat -r 200 --mate-std-dev 200 -a 8 -m 0 -I 1000000 -p 4 -g 100 -o $OUTDIR/ --solexa1.3-quals --coverage-search --microexon-search --max-segment-intron 1000000 -G $GFF3 $INDEXDIR/hg19 $READDIR/s_1.fq $READDIR/s_2.fq
    "

    By the way, the FLAG (second field) in the .sam file is not correct in this case:
    163="10100011"
    83="1010011"
    This means "The read is mapped in a proper pair", but they are on the different chromosome, how can they be properly paired?

  • #2
    Can you construct a small dataset I can use to reproduce this issue? It's probably a bug in matching up the left and right reads from each pair, and I haven't seen it before. If you email me a test set, I'll run it through the debugger. Thanks for your feedback.

    Comment


    • #3
      I have send you the email, please check it~~

      Comment


      • #4
        Hi,

        I was wondering if this is a recognised bug?

        Well actually two questions:

        (1) should TopHat be mapping mate pairs to separate chromosomes in the first place?
        (2) if this is allowed, should the MRNM field have a different value?

        Thanks for your time.
        Last edited by Bio.X2Y; 05-28-2010, 03:42 AM. Reason: typo

        Comment


        • #5
          I have the same problem too, and here are several examples from my SAM file.

          GAII04_0001:1:118:9134:2400#0 147 chr1 14092 255 36M = 14197 0 GAGCAAACTCCAAGACACCTTCTACCCCGACACCAG CCCCCCDCCCCCCCCCCCCCCCC@CCCCCCCCCCCC NM:i:2
          GAII04_0001:1:118:9134:2400#0 99 chr9 14197 255 36M = 14092 0 AACCACTTGAGCAAACTCCAAGACACCTTCTACCCC CCCCCCCCCCCCCCA=CCCCDCDCCCC@CCCBBDCD NM:i:1
          GAII04_0001:1:119:10145:4447#0 163 chr1 18372 255 36M = 18561 0 GGGAGATCCTGCCATGGAGAAGATCACAGAGGCTGG CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC NM:i:0
          GAII04_0001:1:119:10145:4447#0 83 chr9 18561 255 36M = 18372 0 AGAGCTGCCAGGCCAGGCCCTCAGGCAAGGGCTCTG DC?CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC NM:i:0
          GAII04_0001:1:17:2127:14575#0 147 chr19 40331067 255 36M = 40331078 0 TGGCTCCACCATCACATTCTTCCCCGACTGGTTTCC CCCCDCCDCCCCCDCCCCCCCCCCCCCCCCCCCCCC NM:i:1
          GAII04_0001:1:17:2127:14575#0 99 chr15 40331078 255 36M = 40331067 0 CTTCTTCAAGGTGATATAGACGCTGCCCGACGTCCG CCCCCCCCCCCBCCCCCCCCCCCCCCCCCCCCDCCC NM:i:0
          GAII04_0001:1:94:17511:16302#0 147 chr6 42048427 255 36M = 42048548 0 CTCTTATAATTTTGTTCTGAGGGGAGACAGGGAAAG CCCCCCCCBBCCCCCCCCCCCCCCCCCCCCCCCCCC NM:i:0
          GAII04_0001:1:94:17511:16302#0 99 chr1 42048548 255 36M = 42048427 0 TCCCTTTTGTTTTCAAACCCTTCTTGGTCTTTTTGG CCCCCCCCCCBBBBCCCCCCCCCCCCCCCCCCCBBC NM:i:1

          For some reason, most of the reads that have this error are mapped to chr1 and chr9.

          The commands that I used to run TopHat and the corresponding reads in the sequence files are as follows:

          tophat --solexa1.3-quals -p 4 -r 170 /databank/indices/bowtie/GRCh37/GRCh37 s_1_1_sequence.txt s_1_2_sequence.txt
          --
          @GAII04_0001:1:118:9134:2400#0/1
          AACCACTTGAGCAAACTCCAAGACACCTTCTACCCC
          +GAII04_0001:1:118:9134:2400#0/1
          bbbbbbbbbbbbbb`\bbbbcbcbbbb_bbbaacbc
          @GAII04_0001:1:119:10145:4447#0/1
          CAGAGCCCTTGCCTGAGGGCCTGGCCTGGCAGCTCT
          +GAII04_0001:1:119:10145:4447#0/1
          bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb^bc
          @GAII04_0001:1:17:2127:14575#0/1
          CTTCTTCAAGGTGATATAGACGCTGCCCGACGTCCG
          +GAII04_0001:1:17:2127:14575#0/1
          bbbbbbbbbbbabbbbbbbbbbbbbbbbbbbbcbbb
          @GAII04_0001:1:94:17511:16302#0/1
          TCCCTTTTGTTTTCAAACCCTTCTTGGTCTTTTTGG
          +GAII04_0001:1:94:17511:16302#0/1
          bbbbbbbbbbaaaabbbbbbbbbbbbbbbbbbbaab
          --
          @GAII04_0001:1:118:9134:2400#0/2
          CTGGTGTCGGGGTAGAAGGTGTCTTGGAGTTTGCTC
          +GAII04_0001:1:118:9134:2400#0/2
          bbbbbbbbbbbb_bbbbbbbbbbbbbbbbcbbbbbb
          @GAII04_0001:1:119:10145:4447#0/2
          GGGAGATCCTGCCATGGAGAAGATCACAGAGGCTGG
          +GAII04_0001:1:119:10145:4447#0/2
          bbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbbb
          @GAII04_0001:1:17:2127:14575#0/2
          GGAAACCAGTCGGGGAAGAATGTGATGGTGGAGCCA
          +GAII04_0001:1:17:2127:14575#0/2
          bbbbbbbbbbbbbbbbbbbbbbcbbbbbcbbcbbbb
          @GAII04_0001:1:94:17511:16302#0/2
          CTTTCCCTGTCTCCCCTCAGAACAAAATTATAAGAG
          +GAII04_0001:1:94:17511:16302#0/2
          bbbbbbbbbbbbbbbbbbbbbbbbbbaabbbbbbbb

          Comment

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