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Old 08-04-2021, 04:21 AM   #1
kstl20
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Default run failed to generate the index reads

Hi everyone,

I recently did a sequencing run with a 600 cycle V3 cartridge on a MiSeq. The run completed but for some reason failed to generate the fastq files. Basically, the run failed to generate the index reads and subsequent read 2 sequences.

After the initial few cycles, the run looked balanced, and the cluster density was ok. The area of concern was after ~260bp of read 1 when the sequencing intensity appears to dip indicating a problem after this point. The run completely failed by ~280 bp of read 1.

All of the subsequent instrument diagnostic tests carried out passed without issue and I couldn't see an issue with my library preparation. I included all of the correct p5, p7 flow cell adapters and Illumina indexes.

Could this be a one of instrument blip? I have been afraid to repeat the run since.

Does anyone have any suggestions or any ideas of why my run could have failed?

Thank you for your help.
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Old 08-04-2021, 04:26 AM   #2
GenoMax
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You should contact Illumina tech support so they can look at the sequencer/run remotely to diagnose the problem. It could be an instrument blip but if there was a problem they can identify it.
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Old 08-04-2021, 04:28 AM   #3
kstl20
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My colleague did. Illumina tech support were unable to determine the exact nature of the problem. Any other suggestions?
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Old 08-04-2021, 05:07 AM   #4
GenoMax
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Then you have no option but to try another run. What % of phiX was used for first run. Did that data look ok?
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Old 08-04-2021, 05:16 AM   #5
kstl20
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We used 5% PhiX and no Phix data was produced from the run. The whole thing failed at base 280 of read 1.
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Old 08-04-2021, 07:53 AM   #6
GenoMax
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You should be able to recover the data for 270-odd cycles and then take a look at it. phiX data should be in there. This may need to be done offline using bcl2fastq on the command line. Not sure if that is an option for you.
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Old 08-06-2021, 02:01 AM   #7
torben
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Quote:
Originally Posted by GenoMax View Post
You should be able to recover the data for 270-odd cycles and then take a look at it. phiX data should be in there. This may need to be done offline using bcl2fastq on the command line. Not sure if that is an option for you.
You can recover the data on the MiSeq too.
If you're using Local Run Manager:
https://emea.support.illumina.com/bu...plete-run.html
For MiSeq Reporter:
https://emea.support.illumina.com/bu...-reporter.html
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