MiSeq Sequencing Short Amplicons

[cement_head]

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

[nucacidhunter]

Two effects:

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

[thermophile]

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