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  • TopHat: the results confused me

    I used TopHat to find exon-exon junctions.

    But the results in the output 'junctions.bed' files confused me.
    I seperated RNA-seq data into two datasets as one is 76bp*2 another is 40bp*2, then ran TopHat individually.

    However, the two 'junctions.bed' files gave different results.
    Here are examples of the two 'junctions.bed' result:
    One reports the following junctions:
    chromosome12 12302 12721 JUNC00000002 5 -
    chromosome12 33389 34997 JUNC00000003 6 +
    chromosome12 33688 34964 JUNC00000004 2 +
    chromosome12 35474 35675 JUNC00000005 5 +
    chromosome12 35718 35949 JUNC00000006 9 +

    Another reports the following junctions:
    chromosome12 12303 12723 JUNC00000005 26 -
    chromosome12 33679 34982 JUNC00000007 3 +
    chromosome12 35490 35674 JUNC00000008 6 +
    chromosome12 35711 35949 JUNC00000009 7 +

    These junction locations of each output file were similar but different.
    When I extracted the detailed sequences, no GT-AG was found.

    Does anybody know how to explain it?

  • #2
    Maria,

    Since you did not give the complete junctions.bed line, I attached two lines
    from my TopHat analysis:

    75nt run
    chr20 251862 256677 JUNC00000001 11 - 251862 256677 255,0,0 2 46,69 0,4746


    50nt run
    chr20 251879 257723 JUNC00000001 2 - 251879 257723 255,0,0 2 29,39 0,5805


    Here is the link to BED format at ucsc: http://genome.ucsc.edu/FAQ/FAQformat.html#format1

    You can calculate the (start,end) of the junction by:
    e.g.for 75nt run, find col 2, 10, 11, 12:
    col 2: feature start 251862
    col 10: block count 2
    col 11: block sizes 46,69
    col 12: block starts 0,4746
    You can find the splice junction coordinates by:
    start: col2+col11.first.item+1
    end: col2+col12.second.item,
    i.e.,
    start: 251862+46+1=251909, end: 251862+4746=256608
    chr20:251909-256608


    for 50nt run,
    junction is : chr20:251909-257684


    Now, in this case, TopHat finds two different junctions. You can also
    copy and paste the BED directly to ucsc custom track and visualize them.

    Hope it helps.

    Lifeng

    Comment


    • #3
      Hi, Lifeng,

      Thank you so much for you kindness.
      I'll calculate these junctions again according to your remind.

      Comment

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