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Old 03-02-2019, 09:14 AM   #1
Junior Member
Location: Cambridge

Join Date: May 2015
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Default Poor per tile quality on top surface

Dear All,
We've been sequencing CRISPR guide RNA libraries for a while and had several high quality libraries sequenced on HiSeq4000. However the latest one shows this QC fail with very poor sequence across almost all of the top surface tiles. We repeated the sequencing on another flow cell and saw exactly the same result.

We add a custom sequencing primer, but have used the exact same library construction and sequencing protocol multiple times before.

I would be extremely grateful if anyone has any helpful suggestions what could be causing this?

Picture attached.

Screenshot 2019-03-02 at 18.06.44 copy.jpg is offline   Reply With Quote
Old 03-03-2019, 04:54 AM   #2
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Location: East Coast USA

Join Date: Feb 2008
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Have you consulted Illumina tech support? You are not doing something totally custom that is unsupported by Illumina?
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Old 03-04-2019, 05:44 PM   #3
Location: Livermore, CA

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You likely lost focus of the top surface. Check the FWHM on top vs. bottom surface for the run. I know some have had intermittent issues with losing focus on this platform when there's a read (even prior) that has zero base diversity. HiSeq 4000 doesn't always recover from this.
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