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Old 03-13-2018, 11:22 AM   #1
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Location: CT

Join Date: Apr 2015
Posts: 243
Default miseq clustering higher bottom than top of flow cell

What can cause different clustering levels on the top vs bottom of the flow cell. I run amplicons, so undercluster generally aim for ~750k clusters. I have a library that I've run twice, both times I'm getting ~700k clusters on the bottom and 200-300k clusters on the top. I looked at the thumbnails since overclustering can lead to poor estimation of cluster density. I really did undercluster-the bottom looks about the same density as I usually get and the top really has about half as much as normal.

library details. 50% bacterial 16s, 30% fungal ITS, 20% phiX. First run 35% PF, 50% phiX aligned, run failed part way through I1. Second run 42% PF, 33% aligned, sample distribution between 16s and ITS is roughly what I expected. Identified top ~40%, bottom ~15%. The 2 runs were on different MiSeq.
Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

Last edited by thermophile; 03-13-2018 at 01:27 PM.
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