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  • 'mapped to different chr' reads in sam flag from TopHat mapping

    I used Tophat to map a RNA-seq paired-end data set and the samtools flagstat gives the following summary:

    Code:
    63211490 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    63211490 + 0 mapped (100.00%:nan%)
    63211490 + 0 paired in sequencing
    32750906 + 0 read1
    30460584 + 0 read2
    53967250 + 0 properly paired (85.38%:nan%)
    58218754 + 0 with itself and mate mapped
    4992736 + 0 singletons (7.90%:nan%)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)
    My question is why tophat gives me '0 + 0 with mate mapped to a different chr' ? I also used BWA to map the same data and it gives '1417632 + 0 with mate mapped to a different chr'. will Tophat ignore the pairs mapped with both end mapped to different chrs or there is something wrong with my settings for running tophat command?

    here is my command:
    Code:
    tophat -r 200 -p 4 -o RNA_seq seq1.fa seq2.fa
    The fragment length is 400bp, and reads are 100bp.

  • #2
    This is a VERY frustrating feature of Tophat as it will not align read pairs to two different chromosomes so there are no such reads in the sam/bam file. I'm still not clear if both don't map or only the second read, my suspicion is its only the second read that doesn't get mapped.

    Tophat fusion or Chimerascan will do this if you are looking for gene fusions / hybrid transcripts

    Comment


    • #3
      Hello there,

      I also have been annoyed with such Tophat's features for a long time, but after tophat2 was released, it has a new option called "report-discordant-pair-alignments" which allows mate pairs to map on different chromosomes.

      I don't know, however, how these mate pairs are treated when FPKM calculating...

      zun

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