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Old 06-01-2018, 04:56 AM   #2
Location: Canada

Join Date: Jun 2013
Posts: 56

Hi Sean,

Aside from PhiX, are you taking any measures to increase diversity in your amplicon libraries such as staggering/offsetting and/or adding other loci?

At a glance, your cluster densities do not appear to be too high but if you have access to SAV (Sequence Analysis Viewer) for you run it might help if you posted the figures of %base/per cycle, density charts of the flow cell, and the density box plots. If not, the "per tile sequence quality" graph from FastQC might help show if there is differential quality over the length of the flow cell. Your amplicon run might not have been overclustered per se, but it might have been overclustered for a low-diversity run and 10% or more PhiX might not have been enough.

Here is a document on Miseq Overclustering in case you find it helpful:

My guess is that your nano Mobio/Qiagen run was of whole-genome libraries rather than amplicons and may have worked better due to their high diversity.

I have seen people get lower PF% (~70%) with single amplicon sequencing despite moderate PhiX spike-in (~15-20%) if they do not stagger/offset their read 1 locus-specific primers.
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