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Old 10-12-2019, 05:25 PM   #1
wei210
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Location: Texas

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Default am I overclustered -- MiSeq

Hello, hope everybody had a good weekend so far. Sorry, I had a naive question and would appreciated it for any help.

I run a PE V2 run for MiSeq (cancer custom lib with Illlumina P7/P5 primer).
My target cluster density 1000-1200k, and I got 1328k, PF 95% and Q30 93%.

Although most of the Thumbnails seems OK (like #1), i did see lots of Thumbnails like #2 and #3 below. Will #2 suggest overclustered and #3 not focused Thumbnails? also, I could not explain why the base C has significant higher density than others (please see picture below) while I think my sample of high diversity. Am I overclusterd? Any explanation to help me understood these will be much appreciated.

Thanks
Wei210
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Old 10-12-2019, 06:55 PM   #2
nucacidhunter
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Default

Cluster density is above specification but final results for PF and Q30 looks good. Those thumbnails are specific for selected cycle and base and if you select different base at the same cycle it will look different depending on frequency. Intensity plot also indicates that library is not as balanced as thought. If you select %base from drop down menu in that plot it will give good indication of base frequency at each cycle.
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Old 10-12-2019, 07:21 PM   #3
wei210
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Thank you so much, that helps lot.
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