Agilent Pre-Capture Pooling with XT library?

[bwubb]

Hello all,

So my lab has done pre-capture pooling using Agilent SureSelect bait libraries before, but that was before they made XT and XT2 kits. We are entertaining the idea of performing pre-capture pooling using and XT library, but with an XT2 reagent kit. I have yet to hear of anyone trying this.

My only caution is I do not know enough about the differences between XT and XT2 (ie. post-capture indexing library and pre-capture indexing library).

If it is just a matter of bait concentration then things should work out ok, assuming we do not multiplex too deeply. I think the max baits to give are 16 anyway.

Anyone have any information/thoughts/experience they could share?

Thanks.

[Richard_Dillon]

Hey did you try this and did it work? Thanks! richard

[bwubb]

Still working on it. Will definitely update with the level of success.

[Gopo]

bwubb - Curious if you could share your results. Did you have to add 3x more XT bait library volume in the hybridization reactions with XT2 reagents (i.e., 15uL baits instead of 5uL) or have to concentrate your baits 3x?

[Gopo]

superduper - thanks for the info! The same group has a homebrew recipe for an AMpure XP substitute http://protocols-serapure.readthedocs.org/en/latest/

[Gopo]

superduper - did you use XT blockers with the XT hyb mix and get okay results? I ask because XT2 adapters contain 8bp indexes while XT adapters have 6bp indices, so both kits would presumably use different blockers.

[biochemist87]

xGen blockers

Hey, has anyone used xGen blockers (Standard or Universal), and have high duplication issues when coming off of their MiSeq? I'm using Nimblegen Capture, with custom capture probes. We used to use Uracil-blockers, and have recently changed to these new xGen blockers, and as I said, are seeing high duplication rates. Any advice would help, thank you!

[bwubb]

Sorry everyone for not updating this thread. I just realized people were waiting for this answer. Unfortunately I do not have one as our lab switched to NEB Next library prep before I could even pilot this reagent scheme. Again Im sorry for the lack of reply.