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Old 01-27-2020, 05:33 AM   #4
martin2801
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Location: Austria

Join Date: Jan 2020
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Quote:
Originally Posted by JoeKutch View Post
We had this issue when trying to sequence 10x libraries alongside 150/100 PE samples on our HiSeq 4000. I spoke to Illumina and they weren't able to give me a clear answer, but basically said that after the intensity drop after the first 28 cycles the focus point drifts. It refocuses during the indexing cycles but then moves back to focus point for R1 (which is now out of focus) for R2 without refocusing. I think the bottom surface focuses better because it's a fixed distance from there to the stage, but the top surface is a little more variable.

This is how I understood it from my conversation, but at this point Illumina didn't really have much knowledge of the issue - if you access to support they may have more and clearer information now.
This actually makes a sense. Yeah we tried to talk to people at Illumina and 10x and none of them could really tell us whats going on or how to fix this issue except for not sequencing dual indexed. Thanks for your explanation!
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