Exome sequencing with Illumina Kit

[lingfung.tang]

I was preparing the 2nd hybridization step of an exome sequencing project. But I mixed up the target elution buffer and the target capture buffer, and used the wrong one (i.e. I used the elution buffer to do the capture). In trying to get back the captured DNA sample, I extracted it with AMPure Beads and run a DNA 1000 chip with the sample. Unfortunately, from the result, I could not find any DNA > 200bp. There is only one sharp band with 20ng/ul at 100-150bp. I suppose that is the target capture oligo I put in the reaction for 2nd hybridization. I also saved the 'wash waste' from AMPure Beads, and found no DNA in it with DNA 1000 chips. I do not understand why AMPure Beads can save the oligos but not the captured library. Do you know if I have any chance I can find back my capture library?

On the other hand, I continue the 2nd hyb with the DNA I extracted from AMPure Beads, are they safe to be kept at -20C before the wash?

Thanks!

[csoong]

I never worked with Illumina's exome kit (only Sureselect) but sounds like the capture step failed due to the elution buffer, the bait never binded with the DNA targets. Or if it did, the AMPURE step failed due to the initial presence of elution buffer.

Also, it's hard to imagine how AMPURE beads could pull down any DNA with elution buffer mixed materials to start with. Just my thoughts may be wrong.

[lingfung.tang]

Thanks. But the AMPure Bead can pull down the target capture oligos

[csoong]

With Sureselect after the capture step there is a magentic streptavidin-biotin pull down I think that might be a step you had run trouble into (if Illumina also has a streptavidin-biotin pull magnetic bead pull down).
Do you think that's the case?

[lingfung.tang]

possible. but how things can go wrong with that case?

[csoong]

my guess is the elution buffer cause the DNA to dissolve in solution instead of binding with the biotinylated baits. So AMPURE only purified the magnetic beads that contained the baits as you thought. But then the question is why are the baits being purified? They are certainly not just single stranded DNA. They are supposed to be biotinylated RNA right? would it still end up as a single band around 150 bp? I do not know...