run failed to generate the index reads

[kstl20]

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.

[GenoMax]

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.

[kstl20]

My colleague did. Illumina tech support were unable to determine the exact nature of the problem. Any other suggestions?

[GenoMax]

Then you have no option but to try another run. What % of phiX was used for first run. Did that data look ok?

[kstl20]

We used 5% PhiX and no Phix data was produced from the run. The whole thing failed at base 280 of read 1.

[GenoMax]

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.

[torben]

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/bulletins/2019/09/local-run-manager--generate-fastq-files-from-an-incomplete-run.html

For MiSeq Reporter:

https://emea.support.illumina.com/bulletins/2016/10/generate-fastq-from-an-incomplete-run-in-miseq-reporter.html