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Old 03-14-2018, 10:49 AM   #2
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Location: Purdue University, West Lafayette, Indiana

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Originally Posted by thermophile View Post
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.
Up to this point I'm just thinking "fluidics issue". But you go on to write:

Originally Posted by thermophile View Post
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.
I mean even running the same pool on two MiSeqs could just mean both MiSeq have the same issue -- like a biofilm starting to mess up the fluidics.

But since you did get through R1 the first time, but errored out part way through indexing, I'm surprised. Was it a focus issue? I mean it has to be that the cluster density of i7 producing clusters dropped too low. But that usually seems to happen during the first index cycle.

I'm going to say this isn't one problem it is two. No library issue should cause different cluster densities on the top vs. the bottom of the flowcell--not unless you are doing something crazy like 1/2 filling the sample slot with mineral oil or something else that could cause a top to bottom density gradient.

So, biofilm + some other library issue.

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