Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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

          Latest Articles

          Collapse

          • seqadmin
            Choosing Between NGS and qPCR
            by seqadmin



            Next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) are essential techniques for investigating the genome, transcriptome, and epigenome. In many cases, choosing the appropriate technique is straightforward, but in others, it can be more challenging to determine the most effective option. A simple distinction is that smaller, more focused projects are typically better suited for qPCR, while larger, more complex datasets benefit from NGS. However,...
            10-18-2024, 07:11 AM
          • seqadmin
            Non-Coding RNA Research and Technologies
            by seqadmin




            Non-coding RNAs (ncRNAs) do not code for proteins but play important roles in numerous cellular processes including gene silencing, developmental pathways, and more. There are numerous types including microRNA (miRNA), long ncRNA (lncRNA), circular RNA (circRNA), and more. In this article, we discuss innovative ncRNA research and explore recent technological advancements that improve the study of ncRNAs.

            Nobel Prize for MicroRNA Discovery
            This week,...
            10-07-2024, 08:07 AM

          ad_right_rmr

          Collapse

          News

          Collapse

          Topics Statistics Last Post
          Started by seqadmin, Yesterday, 06:09 AM
          0 responses
          10 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 10-30-2024, 05:31 AM
          0 responses
          13 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 10-24-2024, 06:58 AM
          0 responses
          22 views
          0 likes
          Last Post seqadmin  
          Started by seqadmin, 10-23-2024, 08:43 AM
          0 responses
          52 views
          0 likes
          Last Post seqadmin  
          Working...
          X