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-   -   Low QC scores? (http://seqanswers.com/forums/showthread.php?t=72654)

ox_zoo 11-18-2016 12:56 PM

Low QC scores?
 
I have just set up my very first sequencing experiment on the Next-Seq 500. It is now 45 cycles into PE sequencing, however it seems that my QC scores are a bit low. I only have a % >= Q30%: 25.2%

I have around:
280 million reads in the QC 10 bin.
25 million in the QC 20 bin
8 million in the QC 25 bin
65 million in QC 30 bin
44 million in QC 35 bin.

Is this a bad result? Apologies for my stupidness, just was wondering whether data could be salvaged from this?

ox_zoo 11-19-2016 05:12 AM

Yes, now it has finished and the QC is only 15.2.

The machine also has a warning message on two of the lanes:

camera disabled:failed to detect clusters


Does this mean that the cameras are not working properly?

Thanks

GenoMax 11-19-2016 06:29 AM

Probably. You should contact Illumina tech support. They can take a look at your sequencer remotely to diagnose the problem.

If your instrument is not directly accessible over the network then you will need to send them some diagnostic files.

ox_zoo 11-19-2016 06:37 AM

Thanks for your reply.

Yes I will do that - unfortunately since its a weekend they cannot reply.

I was thinking - if clusters are not formed in those two particular areas, would the cameras be disabled? My pooled library concentration was sufficient, so I did not anticipate any problems. Also, if the cluster density is low then I am not sure how QC would be affected..

Many thanks

GenoMax 11-19-2016 06:42 AM

What was the cluster density for this run?

If the run was borderline overclustered to begin with, it is possible that sequencer lost the ability to distinguish the clusters over time (they become fatter). There is also the possibility of this being some sort of a hardware issue.

ox_zoo 11-19-2016 06:48 AM

The cluster density for the four lanes range from 0-250k/mm2.

The lanes:
Lane 1: 235 41
Lane 2: 237 38
Lane 3: 212 56
Lane 4: 230 35

I hope this helps.

GenoMax 11-19-2016 07:50 AM

This run is borderline overclustered (as I recollect for NextSeq optimal cluster density is 170-220 K cluster/mm^2).

Since there is nothing to lose at this time contact tech support and see what they say. You may have to eat the cost on this one and re-run.


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