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Old 09-16-2013, 05:32 AM   #1
megsmcbride
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Default MiSeq Error stopped run

I started a run on the MiSeq on Friday and by Monday morning I noticed that an error caused the MiSeq to stop just less than 2 hrs into the run. The error saids "No useable signal found in the images; it is possible that clustering has failed." Has anyone recieved this error before? I was running a few Nextera XT libraries. I have used this procedure before with success. This error is a first.
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Old 09-16-2013, 06:11 AM   #2
GenoMax
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Have you checked images in the "Thumbnail_Images/L001/C1.1" directory to see if there are clusters?
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Old 09-16-2013, 06:20 AM   #3
NextGenSeq
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If you spike in PhiX you will be able tell if it's a chemistry or instrument error. Without spiking it is difficult to troubleshoot.
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Old 09-16-2013, 06:32 AM   #4
megsmcbride
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No thumbnail images were created and I didn't use PhiX. What I can tell you is that the machine went under rountine maintenance and a replacement of the motor that controls the focus mirror. The machine worked very well after the service job, but we used a non-expired flowcell and reagents. For this run we used an expired flowcell (june 6th expiry) and expired reagent cartridge. Do you think that the expired flowcell may have caused this level of failure?
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Old 09-16-2013, 06:46 AM   #5
GenoMax
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It may be best to open a ticket with Illumina tech support so they can remote-in to look at this run.

If there was a hardware failure it should show up in the "Logs" directory. You could look through those files (*.log) while you wait to hear back from Illumina.

Expired kit (unless it was mishandled in terms of storage) should not cause a total failure.
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Old 09-16-2013, 06:54 AM   #6
mcnelson.phd
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The reagents should still have been fine even though they were "expired".

I had this problem once with a run, and the issue was ultimately chalked up to a "bad flowcell". That excuse gets thrown around a bit by Illumina when they don't have any other explanation, but they'll usually replace the kit so you can re-run the libraries.

As was noted above, you should always have phiX spiked into your samples so you can troubleshoot more effectively. Right now you don't know if your libraries or the kit or the MiSeq were at fault, although my suspicion would be the libraries.

Did you do any library QC before trying to run them or did you just follow through the XT protocol? We use XT extensively (no pun intended), and we've had issues when following the denaturing and normalization parts of the protocol. We've since switched to stopping after the post-PCR cleanup and doing Qubit and Bioanalyzer to QC the library, followed by standard pooling and denaturing like these were standard libraries. Since we've switched to doing things this way, we've been getting more even pooling and have more control over clustering.
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Old 09-16-2013, 10:12 AM   #7
megsmcbride
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I contacted Illumina and they had me run a number of diagnostic tests to make sure there was nothing wrong with the MiSeq. All tests passed. Therefore I was told that these errors are caused by the presence of bubbles at the wrong place and time. For example, a bubble present near the inlet port, where the optical system attempts to find focus, of the FC can cause focusing problems and lead to a premature run stop. Apparently, these errors do occur from time to time.

Thanks everyone for your help.
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