Low QC scores?

ox_zoo · 11-18-2016, 12:56 PM

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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11-18-2016, 12:56 PM	#1
ox_zoo Junior Member Location: Oxford Join Date: Nov 2016 Posts: 5	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?

11-19-2016, 06:37 AM	#4
ox_zoo Junior Member Location: Oxford Join Date: Nov 2016 Posts: 5	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 Last edited by ox_zoo; 11-19-2016 at 06:39 AM.

11-19-2016, 05:12 AM	#2
ox_zoo Junior Member Location: Oxford Join Date: Nov 2016 Posts: 5	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

11-19-2016, 06:29 AM	#3
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 6,698	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.

11-19-2016, 06:42 AM	#5
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 6,698	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.

11-19-2016, 06:48 AM	#6
ox_zoo Junior Member Location: Oxford Join Date: Nov 2016 Posts: 5	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.

11-19-2016, 07:50 AM	#7
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 6,698	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.