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Old 12-16-2017, 04:27 AM   #1
cement_head
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Question 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
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Old 12-16-2017, 07:25 PM   #2
nucacidhunter
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Two effects:

1- As noted wasting sequencing
2- Low overall Q30 score for the run without affecting inserts Q scores
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Old 12-18-2017, 03:09 PM   #3
thermophile
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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
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