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Old 01-21-2020, 09:20 AM   #2
Location: Philadelphia

Join Date: Oct 2016
Posts: 17

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.
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