Miseq Error: No usable signal found in the images; it is possible that clustering has

[rlopez0218]

Hello all,

I wanted to reach out to see if anyone has experienced this error, descript below. The run appears to have failed shortly after starting it and did not take any images of clustering. I was paranoid that the sample may not have denatured optimally before adding the HT1 buffer to neutralize the solution, even if this was the case I feel SOME clustering could have occurred.

Error:
"No usable signal found in the images; it is possible that clustering has failed"

[microgirl123]

The best thing to do in cases like this is contact Illumina tech support. They can have a look at the instrument logs and see what's happened!

[Jessica_L]

I just had this happen over the weekend, too. A run I had set up on Friday failed with the same message. Am working with Illumina tech support-- have already done the systems check and that all passed. I'm going to go re-qPCR my sample libraries just to be sure those are all fine, but even the 10% PhiX spike-in that we were using didn't work.

[rlopez0218]

Just did a systems check and found that the flow cell I used didn't pass the volumetric test. I then post run washed the thing then did the volumetric test with a previously used flow cell from last week. It passed. I'm going to try my sample again

[Jessica_L]

Mine appears, Illumina believes, to be a hardware issue. They're sending out an FAS to check things out.

[jlodil]

same error in 9/30/15 run

Hello I am experiencing the same issues with my current miseq run using the trusight tumor panel on a 600 cycle v3 kit. My run also terminated shortly after commencing even though the screen reflected that the run had completed but with errors . I am now conducting a volume test and am contacting illumina support for guidance. Any response or solutions you guys get I would appreciate to troubleshoot my issues.

[crioceris]

Hi, I have the same error. What illumina said you about?

[GenoMax]

Hi, I have the same error. What illumina said you about?

Best recourse is to contact Illumina tech support. They can take a look at your machine remotely (or ask you to send some files if the machine is not on the network). This error can be because of both hardware and/or library issues.

[AlexT]

we had the same issues, illumina had no idea what went wrong; first we were told to get replacements, afterwards they wanted to do one million other tests
until now, we did not get a replacement nor any new information; we have problems with the 600cycle kits from the beginning but illumina still denies to admit that they have a problem with the kits (since we have succesfully performed more than 400 500cycle runs it is probably not our problem)
we therefore won't use the 600cycle kit anymore after the current project

[GenoMax]

@AlexT: Are you saying that error described in this thread is what you saw with 600 cycle kits?

There seems to be an ongoing issue with Q-scores and 600 cycle MiSeq kits but we have never had the error that is the subject of this thread occur with those kits.

[AlexT]

@GenoMax: I cannot remember the exact error message, but we had once 0 clusters and once only a few despite using >20% PhiX; for testing we spiked in these samples into a 500 cycle run and they performed perfectly

Q-problems are our usual friends with the 600 cycle kits

[Khillo81]

If I may ask, what concentration of NaOH did you use to denature the library? How did you prepare the dilution?

[crioceris]

Hi all,
Illumina checked instrument and give me new kit. It worked fine. I was not sure about, because it was our first run. They don´t give me explanation what happened first time.

to khillo81: I prepared 1M solution and diluted it to 0,2M.

[Khillo81]

Well, problem solved it seems. Usually, when clustering fails completely one reason could be the NaOH which is why Illumina recommends using a fresh dilution every time. NaOH reacts with CO2 in the air to give NaHCO3. Using higher concentrations of NaOH and keeping them in tightly sealed bottles is very important. We use a stock of 10N NaOH to prepare our 0.2N dilutions and it works great. I have had the same problem as you faced once and after asking a couple of questions, Illumina sent a replacement kit and the run worked. They also did not provide an explanation of why the first run might have failed.