High phasing on GAIIx and HiSeq

[james hadfield]

Has anyone seen high phasing rates recently?

We have seen phasing of over 0.4% and up to almost 0.8% recently on several runs across both GAIIx and HiSeq.

This has been evident in paired end runs where one read is fine but the other is poor.

I believe this could be due to chemistry. I would like to find out if others have experienced similar issues over the past few weeks?

Feel free to contact me directly if you prefer.

[hoisinjl]

When was the last time a maintenance had been done? We've seen these high numbers when either an incorrect control lane was used or it had been too long between maintenance washes.

[james hadfield]

Our instruments always get the required maintenance washes. This is unlikely to be the case as we have seen one read in a pair having higher pahsing than the other where a 200cycle SBS kit is used for the first read and a separate on for the second on a pair of flowcells.

[huguesparri]

We faced a high phasing/prephasing problem once. Illumina's replaced our kits and gave us the following advices:

Here are some guidelines for the future:
Handling properly the reagents is essential for avoiding phasing/prephasing problems.
Therefore the best practices are:
- Store SBS kits with the plastic bagging intact. Avoid warming, shaking or aerate the reagents.
- Keep ICR buffer at 4ºC (or frozen at -20C) and LPR and LFN should be added immediately before loading on the sequencer. ICR should be tightly closed whenever possible and ICR and Incorporation Mix should never be stored at temperatures above 8ºC.
- LFN should be thawed in room temperature water for 20 min; CMR should be thawed in the fridge overnight and thawing should be finished in room temperature water for not more than one hour and, as you know, in a different container as any other reagent.
4) Maintenance Washes (water-NaOH-water) are absolutely essential after each run and the waste volumes from each phase should be checked.