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  • tophat2 mapping PE and SE reads simultaneously

    Hello everyone. I have a single library of ~ 30 million paired end reads. After trimming primer sequences and adaptors (it was a highly amplified library from RNA), about half of my reads become single ended (I lose read 2 which is mostly primers). When I use tophat2 to try and map the PE and orphan SE reads simultaneously I get the following mapping stats.

    Left reads:
    Input : 15693801
    Mapped : 15021980 (95.7% of input)
    of these : 4774963 (31.8%) have multiple alignments (1996947 have >20)
    Right reads:
    Input : 15693801
    Mapped : 14981893 (95.5% of input)
    of these : 4767368 (31.8%) have multiple alignments (1996950 have >20)
    Unpaired reads:
    Input : 12548124
    Mapped : 11939794 (95.2% of input)
    of these : 4816113 (40.3%) have multiple alignments (83 have >20)
    95.5% overall read mapping rate.

    Aligned pairs: 14492245
    of these : 4742324 (32.7%) have multiple alignments
    1998194 (13.8%) are discordant alignments
    79.6% concordant pair alignment rate.

    Then, if i map the paired end reads and single end reads in separate tophat2 runs, I get different mapping stats (the biggest difference is the discordant alignments)

    Left reads:
    Input : 15693801
    Mapped : 15021392 (95.7% of input)
    of these : 4844003 (32.2%) have multiple alignments (2137068 have >20)
    Right reads:
    Input : 15693801
    Mapped : 14981461 (95.5% of input)
    of these : 4836357 (32.3%) have multiple alignments (2137069 have >20)
    95.6% overall read mapping rate.

    Aligned pairs: 14491403
    of these : 4812629 (33.2%) have multiple alignments
    2107050 (14.5%) are discordant alignments
    78.9% concordant pair alignment rate.

    and here are the separate SE stats (also slightly different)

    Reads:
    Input : 12548124
    Mapped : 11937315 (95.1% of input)
    of these: 4800413 (40.2%) have multiple alignments (35 have >20)
    95.1% overall read mapping rate.

    Why should there be any difference if they are mapped in the same run versus separately? (mapped with same reference and same commands and done as per the tophat2 manual by adding the SE reads after a comma to one of the two paired end read files)

    Evan

  • #2
    while the stats for your different methods don't look identical, they aren't all that different.

    Have you tried running one of the methods several times to see how much the results differ from one run to the next?

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