Maximum clustered flow cell life?

[pmiguel]

What is the longest you have waited after clustering a flow cell (especially a PE flow cell) before running it?

I have a PE (v3 chemistry) flow cell that was clustered 4 weeks ago (December 21st, 2011). We thought it defective at the time because edge finding failed every time we attempted to run it. Subsequently we found the edge calling issue was a result of a combination of new edge/autotilt methodology in HCS 1.5.0 and an install failure to update a config file with a parameter critical to the new software.

We plan to re-hybe the 4 week old flow cell tomorrow and attempt to run it. It has been sitting in buffer at 4 oC since December 21st. Hopefully the first base report will tell us whether that is going to be productive or not. I guess I am most concerned that the first read will look okay, but turn around will perform poorly.

Illumina tech support thinks it is worth a shot. Specifications call for a flow cell to be rehybed (sequencing primer re-annealed) after 10 days. But as to whether a flow cell is still functional at 30 days post-clustering, there seems to be no information.
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Phillip

[GW_OK]

Well, unless there's DNase or something in there I don't see why the clusters wouldn't still be there. I mean, aren't they covalently linked to the surface? As long as you're doing a rehyb it should still work. Keep us posted, though...

[pmiguel]

There is some additional chemistry embedded in the oligos on the surface of the flow cell that is used during "turn around". I don't know what that is, or if it "degrades" over time.

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Phillip

[pmiguel]

The First Base Report was marginal. I can see clusters and the focus scores are quite high. But the G and T intensities are mostly near the bottom edge of acceptable.

Decided to run at least until tomorrow. Then recluster if it appears necessary.

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Phillip

[pmiguel]

Read1 (101 nt) completed. Results are acceptable. Got >50 billion >Q30 bases from the flow cell surface. (The HiScanSQ will only image the top of the flow cell.) Index read now in progress. Cautiously optimistic.

Although G signal started very low (just above 1000), it dropped very little during the run. Whereas A and C signal were where one expects them to be, but dropped substantially (as is normal) during the run.

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Phillip

[pmiguel]

Forgot to give the final results: pretty good. The final data set did not appear to suffer much, if at all, from having the flow cell sitting at 4 oC for a month before it was run. Of course, your mileage may vary...

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Phillip