Intensity dropped from cycle 2 dramatically,twice...

[illuminaGA]

Dear everyone.

I need your help.

I started first run 06/28 , the first base was very good ,the intensity was around 600, but from cycle 2 the intensity drop sharply,I only found cluster at bottom of flow cell.Other parts were black.

illumina Tech support advised me to check the fluidics by lane. All lane were good(I run a pre-wash, and collected 2.1ml in each lane)

So I re-Hyb yesterday and re-run the sequencing.

But this happened again.

The intensity was ok in the first base. The intensity was around 400. But from cycle 2,the intensity dropped dramatically.

I attached a picture run2-cycle1-2-2(1), the lane 1-4 finished cycle2,lane 5-8 did't start.

I used the fresh thawed reagents.So the reagents should be ok.

Some one konw what happened here?

Thank you so much!

PS:We run at GAiix

[GenoMax]

Illumina tech support may be your best. They can access your machine remotely (a WebEx type session, if you have not done this before) and try to diagnose the run. Open a ticket with tech support.

[cdalgard]

We are having a very similar issue right now. First base report is outstanding, then we have very very low intensities for Cycle 2 and onward. Only the bottom of the flow cell has intensities. We have checked and validated fluidics, but that is not the issue. We should communicate and compare notes. Thanks!

[illuminaGA]

Hi ,we finished 3 round test running and got same results. We also checked the fluidics taking and collecting ,they were all good. Illumina tech support thought may be something wrong on the temperature control unit or incorrect loading. They will send a FSE to our lab this week or next week.

Are you using GAiix?

Good luck.

We are having a very similar issue right now. First base report is outstanding, then we have very very low intensities for Cycle 2 and onward. Only the bottom of the flow cell has intensities. We have checked and validated fluidics, but that is not the issue. We should communicate and compare notes. Thanks!

[cdalgard]

We completed two rounds of test runs and also got the same poor results (dramatic drop in Cycle 2 intensities). Fluidics post-wash and maintenance run are good and validated. Illumina tech support is still investigating. Your suggestion about temperature control unit (or Peltier cooler) is interesting. I think it is not coincidence that we have the same issue on a GAiix at the same starting time (July 2013). I wonder if we are using the same lot of certain reagents which is causing a new, not previous understood problem. Would you like to compare lot #s of reagents to see if anything matches?

[illuminaGA]

We completed two rounds of test runs and also got the same poor results (dramatic drop in Cycle 2 intensities). Fluidics post-wash and maintenance run are good and validated. Illumina tech support is still investigating. Your suggestion about temperature control unit (or Peltier cooler) is interesting. I think it is not coincidence that we have the same issue on a GAiix at the same starting time (July 2013). I wonder if we are using the same lot of certain reagents which is causing a new, not previous understood problem. Would you like to compare lot #s of reagents to see if anything matches?

Oops,I just trashed all the reagents boxes last Friday. But I have checked the lot No. before each running.They were different. Two of them were our inventory and another one was replacement reagent from illumina.

I got a good running at our other GAiix, and finished last Friday, so We knew that the reagens and flow cell were okay. I loaded all reagents and flowcell form other GA to this one. Guess what. The problems showed again.

I am waiting for FSE, and I hope he can solve this issue.

Did your flow cell charts look same with ours?

[cdalgard]

We also received replacement reagents from Illumina between our two runs and had failures, so that scenario and your scenario suggests it is not reagents.

Can you explain what you exactly mean by "I loaded all reagents and flowcell from other GA to this one."? Does that mean that you took a clustered flowcell and SBS reagents from a completed run on the working GA and transferred them to the bad GA and started a new run after rehyb?

Yes, the flow cell charts look almost exactly the same with yours. We have high intensities on Cycle 1, then they drop to <100 in Cycle 2 and the tile graph shows a gradient of low intensities to higher intensities from top to bottom.

Have you ever had a problem with your Peltier before? We actually had a tuneup of our thermal control in the past when we could not get Resynthesis to complete from Index Read to Read 2. A FSE measured temperatures and calibrated the Peltier, that fixed the problem. I wonder if that system is off again.

[illuminaGA]

Hey,cdalgard
Yes ,you are right. I took a clustered flowcell and SBS reagents from a completed run(PE 101+7+101) on the working GA and transferred them to the bad GA and started a new run, but I didn't rehyb flowcell. That followed the Techsupport's suggestion.

[illuminaGA]

Have you ever had a problem with your Peltier before? We actually had a tuneup of our thermal control in the past when we could not get Resynthesis to complete from Index Read to Read 2. A FSE measured temperatures and calibrated the Peltier, that fixed the problem. I wonder if that system is off again.

I am not sure. Our GAs are used, we brought them from other lab.

[GenoMax]

I am not sure. Our GAs are used, we brought them from other lab.

I hope people who are buying these used GA's are purchasing a maintenance contract (preferably through Illumina or a third party) otherwise ...

[cdalgard]

GenoMax - Our laboratory received a new GAiix and Cluster Station for no cost ($0). We purchased a c-Bot from Illumina directly before GAiix installation to improve our workflow. Illumina honored the original warranty and service contract on the GAiix. They also provided a Dell Precision T5700 to improve the acquisition system. So our GAiix is less than 2 years in operation. However, it was manufactured in 2008 or 2009. That is my concern about failures due to longevity.

[GenoMax]

GenoMax - Our laboratory received a new GAiix and Cluster Station for no cost ($0). We purchased a c-Bot from Illumina directly before GAiix installation to improve our workflow. Illumina honored the original warranty and service contract on the GAiix. They also provided a Dell Precision T5700 to improve the acquisition system. So our GAiix is less than 2 years in operation. However, it was manufactured in 2008 or 2009. That is my concern about failures due to longevity.

I was mainly referring to those who are purchasing used GAIIx's that were traded-in (highly doubt Illumina is making new GAIIx now) as a number of core labs upgraded to bigger instruments.

As with any complex piece of electronics *any* problems (including the computer attached to sequencer) potentially could result in a bill that may be well beyond budgets of small labs, if the instrument is not under a maintenance plan.

If you did not get a maintenance contract it may be a good investment/insurance to plan for and acquire one, especially if the sequencer is an integral part of your projects.

[illuminaGA]

I was mainly referring to those who are purchasing used GAIIx's that were traded-in (highly doubt Illumina is making new GAIIx now) as a number of core labs upgraded to bigger instruments.

As with any complex piece of electronics *any* problems (including the computer attached to sequencer) potentially could result in a bill that may be well beyond budgets of small labs, if the instrument is not under a maintenance plan.

If you did not get a maintenance contract it may be a good investment/insurance to plan for and acquire one, especially if the sequencer is an integral part of your projects.

Hi, I heard form illumina Techsupport that they can provide "golden level contract". The FSE will take care your sequencer. Is this the "maintenance plan"?

[cdalgard]

I agree with GenoMax on having a service contract for integral systems of projects. You just have to have that cost in mind when acquiring a platform. It is alarming to think that sequestration has policy to eliminate new service contracts. It will cost those institutions more funds in the long term.

The Standard vs Silver vs Golden levels of service contract refer to how quickly they will prioritize you for a reported issue. With Golden, they guarantee immediate visit by the FSE. With Standard, they state 5 business day average on-site response time.

[GenoMax]

Hi, I heard form illumina Techsupport that they can provide "golden level contract". The FSE will take care your sequencer. Is this the "maintenance plan"?

Yes. But there are generally several levels available. Ask for all options before deciding what is appropriate for your needs.