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  • Paired-end reads mapped 50% while each pair reads mapped 80%

    Hi all,

    I am new to the RNA seq world. I have processed bunch of illumina paired end reads. The reported alignment is only 49%.. When I use bowtie to map each pair of the paired-end reads, I got 80% of the reads mapped.

    bowtie -n 3 -p 10 --best -e 200 --trim5 15 --trim3 25 --sam BowtieIndexes/mm9 -1 Raw_files/sample-1_export.fq -2 sample-2_export.fq sample.sam


    # reads processed: 21482797
    # reads with at least one reported alignment: 10637665 (49.52%)
    # reads that failed to align: 10845132 (50.48%)
    Reported 10637665 paired-end alignments to 1 output stream(s)


    I was wondering if this is normal. Here's the command and output. Thanks.

    P.S.: This is my 3rd post on this forum. I posted a similar question in my 2nd post. However, I can no longer reply that post.


    ----- I couldn't post a reply, so here it goes ----

    Isn't it that tophat also uses bowtie to align reads?
    Last edited by zack80.liu; 03-02-2011, 03:48 PM.

  • #2
    You shouldn't be using bowtie for RNA-seq (especially with PE reads) - try tophat or another one of the splicing-aware aligners

    Comment


    • #3
      Isn't tophat also use bowtie to do the alignment?

      Comment


      • #4
        Dear Zack,

        The most likely explanation would indeed be that a sizeable fraction of you library fragments are spanning a splice junction. In these cases many fragments in your library are longer than the allowed Bowtie default fragment size of 250bp (These parameters can be adjusted with
        -I/--minins <int> Default: 0.
        -X/--maxins <int> Default: 250)

        Using TopHat or any other splice junction aware aligner should indeed fix your problems.

        Comment

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