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Old 12-12-2019, 03:11 AM   #1
7891_ZA
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Location: Cape Town

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Default NextSeq550_LowClusterDensity_Low%PF?

A 1.8 pM diverse library was loaded on a high-output reagent kit on the NextSeq550 platform.

I understand that an over-clustered run will result in poor signal purity and a low percentage of clusters passing filter (%PF).

But what would cause a severely under-clustered run (~15 K/mm2) to result in a low %PF (60%)?

I have checked the best focus images of this NextSeq550 run, and they definitely appear to be under-clustered, so the reported cluster density does not seem to be '...an underestimation of the true density due to saturation...'.

Furthermore, other than potential issues with sodium hydroxide concentration, quantification, and the actual sequencing primer, what other causes could result in reduced cluster densities?

A phix run had been run on the instrument and achieved optimal clustering thus ruling out any machine related issues.
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Old 12-12-2019, 08:29 AM   #2
Johnwang
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how did you quantify your libs for sequencing ? by my experience, only qPCR method works well for lib quantification. I use illumina lib quantification kit from KAPA.
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low % pf, nextseq550, underclustering

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