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Specify Bowtie2 to align a mate pair only to the same RefSeq

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  • Specify Bowtie2 to align a mate pair only to the same RefSeq

    Hey everyone,

    I am doing exome sequencing of multiple genes, of which some share great homology.
    Some of the read pairs don't share an overlap and thus cannot be merged.
    At the moment I'm aligning to multiple RefSeq's but I realized, that Bowtie2 will align Mate 1 to to gene x and mate 2 to gene y.
    How can I restrict the alignment to align a pair only to one RefSeq ?

    My Ideas which could solve the problem.
    1. On my search for an answer I've stumbled upon this entry (SAM format paired-end alignment handling). Summarized; the answer was that to identify mate pairs mapped to the same RefSeq one should have a look at the 7th (RNEXT) and 8th (PNEXT) column of the sam file. If RNEXT = PNEXT the pair was aligned to the same RefSeq. Thats on how to identify such read alignments. However, is it possible to restrict the alignment such that "RNEXT = PNEXT" holds and it doesn't allow for RNEXT ≠ PNEXT ?
    2. If -X/--maxins <int> is specified to the longest amplicon generated, will Bowtie2 then still seperate the pairs or does implementing this command restrict the alignment to be only on one RefSeq (-> RNEXT = PNEXT)?
    Do you have any idea how to implement this into my alignment code ?
    Thanks in advance for your help
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