NextSeq underclustering

acockburn · 10-16-2019, 07:20 AM

Has anyone had issues with underclustering on the Illumina NextSeq 500/550? We have been having issues all summer. Even increasing input 3 times our normal has not caused overclustering. We usually clustered around 200 K/mm2, now we typically get 100-140 with 3 times the amount. It is not overclustered as we still get good PF and Q30 scores.

We have checked the qPCR, the quant kits, all the reagents, seems the only variable left is the NextSeq.

We think it may have started with a change to the v2.5 Flowcells, but are uncertain.

Anybody experiencing this? Anybody have any suggestions?

Thanks.

Saravanan · 04-15-2020, 10:45 PM

We had issue with low PF few months ago. We called the engineer to fix it. He cleaned the camera and other stuffs and now it's working fine.

jteeee2 · 04-17-2020, 06:35 AM

Could try purchasing some new Sodium Hydroxide and Tris for denaturing libraries. If your NaOH is over a year old and has been opened a lot, I'd recommend purchasing a new bottle. The lenses on the NextSeq get covered in dust over time. Usually a reduction in clustering due to lenses being dirty coincides with a lower PF rate.

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10-16-2019, 07:20 AM	#1
acockburn Member Location: Pa Join Date: Aug 2013 Posts: 10	NextSeq underclustering Has anyone had issues with underclustering on the Illumina NextSeq 500/550? We have been having issues all summer. Even increasing input 3 times our normal has not caused overclustering. We usually clustered around 200 K/mm2, now we typically get 100-140 with 3 times the amount. It is not overclustered as we still get good PF and Q30 scores. We have checked the qPCR, the quant kits, all the reagents, seems the only variable left is the NextSeq. We think it may have started with a change to the v2.5 Flowcells, but are uncertain. Anybody experiencing this? Anybody have any suggestions? Thanks.

04-15-2020, 10:45 PM	#2
Saravanan Junior Member Location: Hong Kong Join Date: May 2017 Posts: 6	We had issue with low PF few months ago. We called the engineer to fix it. He cleaned the camera and other stuffs and now it's working fine.

04-17-2020, 06:35 AM	#3
jteeee2 Member Location: Illinois Join Date: Oct 2014 Posts: 42	Could try purchasing some new Sodium Hydroxide and Tris for denaturing libraries. If your NaOH is over a year old and has been opened a lot, I'd recommend purchasing a new bottle. The lenses on the NextSeq get covered in dust over time. Usually a reduction in clustering due to lenses being dirty coincides with a lower PF rate.