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  • exon capture & mate-pair: Which order?

    I want to create a mate-paired library for exome sequencing using the Illumina platform. Would I exon enrich using a Nimblegen array first and then create the mate-pairs, or would I fragment, circularize, cut to create the mate-pair library first and then filter these for exon regions?

  • #2
    From what I've read, you're going off the beaten path with this. Presumably your goal is to acquire sequence from the neighborhood of your targeted regions.

    You'd get a bigger sampling of the neighborhood by hybridizing first. But, given that these will be much larger fragments than the protocols were developed for, you may find the hybridization conditions require alteration.

    If you create the mate-pairs first, the key question would be are there any recurrent sequences generated by this procedure which need to be blocked and are not covered by the exome kit you are using.

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    • #3
      If you hybridize first, I would be concerned about DNA from your capture reagent making it into your library. It probably wouldn't occur at very high frequency, but depending on you application, it might matter to you.

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      • #4
        Have you considered exome hybridizing with the circularized mate pair library (before cutting)?

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        • #5
          No I hadn't considered that, on the concern that it might lead to less than optimal hybridization and loss of some target regions. But this concern isn't based on any data. Has anyone tried this approach?

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