Do you freeze hyb cocktails?

[jlove]

I asked Illumina about whether or not I could prepare my flowcell hyb cocktails well in advance and freeze them, and the official recommendation was not to store them for longer than a day or two. When I asked specifically why, they couldn't really tell me, but I got the impression that it might be related to loss of yield due to DNA sticking to the tubes, or perhaps instability of the sample in the hyb buffer. However, I could swear I've heard that labs do this regularly.

My question is: In the real world, do labs freeze (or otherwise store) samples in hyb buffer for later use? If so or if not, why? For how long?

Thanks!

[ECO]

Yes, all the time for weeks and months with no severe variation in clustering efficiency. We use eppendorf DNA lo-bind tubes out of paranoia but haven't tested other tubes.

[protist]

We also freeze ours and on occasion I have gone back an reused them for another flowcell.

[TonyBrooks]

We try not to, due to Illumina best practices recommendation. Although I have done a few times and didn't see an impact on data from what we would expect.
Our FAS also suggested we could freeze-thaw our PhiX hyb cocktail to avoid prepping a new one each time.

I suppose in an ideal world someone could do a fresh hyb cocktail vs multiple freeze-thaw from the same aliquot. I would be surprised it Illumina hadn't done this - so there must be some long term impact.

[behavin]

We store our fully diluted hyb cocktails in case of lane failures. I've noticed around a 10% loss of cluster density when we reuse the cocktail however. Recently, instead of reusing the fully dilute HT1+lib, I stored the 20pm tube and then diluted that down to a working concentration about 10% higher than we loaded the first time around. I saw good results from that.

[garyb]

Recently new Illumina user documentation has stated that denatured+diluted 20 pM sample prep libraries can be stored for up to three weeks at -15C to -25C with little or no cluster density reduction. They can be stored for longer lengths of time but a small decrease in cluster density may result. Use of low binding (adsorption) plastic tubes is recommended.

from an Illumina customer support representative

[jlove]

Garyb- do you have a link or can you tell me which user documentation says this? I'd love to have a copy. Thank you!
-Jen

[garyb]

See page 77 of the latest MiSeq User Guide, link below, you will need to be a registered myIllumina member to access documentation.

https://icom.illumina.com/download/summary/uDUWEnvxcE22mfFTC95zwg

These guidelines should eventually make it into the related HiSeq user documentation. The recommendation is good for all Illumina next generation sequencing platforms since they all work on the same Bridging Amplification process inside the flowcell.

from an Illumina customer support representative

[clarkchan]

we freeze ours for a month in lo-bind tubes,not sure much longer