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  • staggered custom sequencing primers to increase diversity

    Has anyone tried using multiple staggered custom sequencing primers in order to increase diversity when amplicon sequencing? I know you can stagger primers in order to generate base diversity. Would using multiple staggered sequencing primers (ie. +1, 0, -1 base position shift) do the same trick or would it introduce a new set of problems? I'll be testing this on a miseq if that makes any difference.

  • #2
    I've discussed this with coworkers and while it should be possible, it adds complexity in the setup/downstream analysis for relatively little benefit since you can also just add more phiX to offset your low diversity. You can get away with some incredibly low diversity libraries (i.e. all same sequence) using a 20% phiX spike-in.

    Here's a paper that describes a similar strategy that is also cool!
    https://bmcmicrobiol.biomedcentral.c...866-015-0450-4

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