[upenn_ngs]

Our lab is preparing to set up a similar seq cap pipeline. I'd be grateful to hear any pros/cons or other thoughts you (or anyone else!) have on this system.

simple workflow and covaris shearing is great. pooling for cost efficiency looks to be tricky as the concentration must be very accurate in order to hybridize multiple sample fragments in one reaction.

[GW_OK]

Does anyone know if you can reuse agilent sureselect once the baits have been captured by the streptavidin beads?

[upenn_ngs]

what exactly do you mean by reuse agilent sureselect? if you are referring to the capture library, then that is a bad idea. the streptavidin beads will pull down all of the biotinylated RNA "baits" whether complementary genomic sequence has been captured or not.

[GW_OK]

But a bound bait could still bind DNA though, no? I think it may be a moot point anyway, as my Agilent rep says the baits are destroyed in an RNase step. But I don't see RNase mentioned in the Sureselect protocol I am looking at...

[upenn_ngs]

I suspect the "bait" RNA is digested during the bead wash steps. Also, Agilent furtively releases updates to the sureselect protocol, so make sure you are following most recent, http://www.opengenomics.com/sureselect

[GW_OK]

It would appear that I have the most updated protocol. The only place I can see RNase being is in the sureselect elution buffer, which I had assumed was merely a NaOH solution given the immediate use of the neutralization buffer afterwards.