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  • MiSeq Sequencing Short Amplicons

    Hello,

    If I am sequencing a heterogeneous mixture of amplicons using a MiSeq (2x250bp) to ensure overlap, and there are some short amplicons (i.e. ~ 150 bp), what happens to the run? Will the rxns simply be wasted on the short amplicons, or will there be adverse affect on the data?

    Thanks,
    Andor

  • #2
    Two effects:

    1- As noted wasting sequencing
    2- Low overall Q30 score for the run without affecting inserts Q scores

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    • #3
      if you have a lot of the shorter amplicons, there's a chance that the software will just kill the run because of difficulty focusing when a lot of the clusters are dark. I've run up to 25% or so short amplicons without killing a run, but never tried higher than that
      Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

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