"Out of focus" on Miseq cluster

[d00b]

Hi Miseq users,

We have low Q score issue with Miseq V3 600 cycles kit.
Cluster density was ~1000K, PhiX was ~5% aligned and PF was higher than 87.8%.

Based on illumina spec, we supposed to have over 70% Q30 score. However, we have very low Q score (67% of Q30). We found this lower Q score was caused by cluster focus issue (we haven't got any error message during the run). We found tile image that contains out-of-focus clusters as following pictures.






Is there anyone has experience on this?

by the way, we have checked phiX run after on-site service. No problem with PhiX test run.

[GenoMax]

What version of MCS are you running? If you are running v.2.6.x, the tech support will likely ask you to downgrade to MCS v.2.5.x. Have you contacted them?

[d00b]

What version of MCS are you running? If you are running v.2.6.x, the tech support will likely ask you to downgrade to MCS v.2.5.x. Have you contacted them?

We have MCS 2.5.05.
Yes, we have contacted them several times and we are still waiting for their review on this run. Do you have same issue with MCS V.2.6.x? Would you share your experience?

Thanks

[GenoMax]

We were running v.2.6.x for a while on several machines and finally hit a "lost focus" error on one of the machines mid-run. At that point advice from tech support was to downgrade to v.2.5. After that downgrade things appear to be working ok (within limits, there is no solution for the Q-score drop for 600 cycle V3 kits).

[Jessica_L]

I know several other users have reported issues with the v3 600 cycle kits, though I don't remember the specifics. I think at least some may have been related to the known MCS v.2.6 issue, but you might try searching around here for more information. I seem to remember several instances of people seeing low Q-scores on 600 cycle v3 kits, unrelated to the MCS v2.6 issue, as GenoMax has said.

How are your 600 cycle runs normally? Do the images look blurry on the left side even early on or does it drift more out of focus as the run progresses? From an SAV/basespace Charts screen, what does the FWHM plot look like?

[d00b]

V3 600 cycles kit's Q score issue has been well noticed.
However, our low Q score issue was also related with focusing problem.
Our normal Q score with V3 600 is over 70% as illumina spec.
Definitely, it is lower than V2 500 cycle kit. FWHM plot is terrible after 250cycles in read1 and 180cycles in read 2.

[GenoMax]

@d00b: The focusing issue sounds specific for your MiSeq. Something an engineer visit should fix.