SMRTbell library prep for BAC DNA to be run on a Sequel

ahay08 · 06-22-2017, 09:25 PM

Hi – just wondering if anyone has done a SMRTbell library prep with BAC DNA? I have to sequence 2 BACs, both approx. 150 kb and I’m having trouble shearing them. I need to make 20 kb libraries but I only have Covaris g-tubes to use for shearing (no access to a Megaruptor unfortunately). The g-tubes are partially successful but I’m still seeing HMW DNA (supercoiled?) DNA bands on my PFG in addition to the 20-40 kb sheared products. I’ve tried linearising them which has helped for one, but the other one has a largely unknown sequence and I haven’t found any restriction enzymes that only cut the vector. Any help would be greatly appreciated, I’m a new Sequel user/library prep technician, and I’m just discovering the intricacies of the various processes!

nucacidhunter · 06-22-2017, 10:08 PM

Possible solutions:
Type I topoisomarase treatment before shearing
Increasing centrifuge speed for g-tube
Pippin size selection of input sheared DNA

Larger fragments may not be an issue for 20kb libraries as they might be lost during insane number of purifications during library prep and they are less likely to interfere with sequencing.

ahay08 · 06-22-2017, 10:35 PM

Thanks nucacidhunter, I'll check out the topoisomerase solution. I tried increasing the centrifuge speed but the tubes tend to get clogged with the HMW DNA so nothing at all passes through. Size selection is a good idea too - normally I do a Blue Pippin 15 kb cutoff for a 20 kb library which collects everything over 15, but I could try defining the size range and see how that goes, thanks. Yes I know what you mean with the purifications - definitely an insane number! I've tried using Ampure beads on the BAC DNA but the beads just clump and they won't resuspend no matter what I do. thanks for your help!

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06-22-2017, 09:25 PM	#1
ahay08 Junior Member Location: Adelaide, South Australia Join Date: Jun 2017 Posts: 2	SMRTbell library prep for BAC DNA to be run on a Sequel Hi – just wondering if anyone has done a SMRTbell library prep with BAC DNA? I have to sequence 2 BACs, both approx. 150 kb and I’m having trouble shearing them. I need to make 20 kb libraries but I only have Covaris g-tubes to use for shearing (no access to a Megaruptor unfortunately). The g-tubes are partially successful but I’m still seeing HMW DNA (supercoiled?) DNA bands on my PFG in addition to the 20-40 kb sheared products. I’ve tried linearising them which has helped for one, but the other one has a largely unknown sequence and I haven’t found any restriction enzymes that only cut the vector. Any help would be greatly appreciated, I’m a new Sequel user/library prep technician, and I’m just discovering the intricacies of the various processes!

06-22-2017, 10:08 PM	#2
nucacidhunter Senior Member Location: Melbourne Join Date: Jan 2013 Posts: 1,166	Possible solutions: Type I topoisomarase treatment before shearing Increasing centrifuge speed for g-tube Pippin size selection of input sheared DNA Larger fragments may not be an issue for 20kb libraries as they might be lost during insane number of purifications during library prep and they are less likely to interfere with sequencing.

06-22-2017, 10:35 PM	#3
ahay08 Junior Member Location: Adelaide, South Australia Join Date: Jun 2017 Posts: 2	Thanks nucacidhunter, I'll check out the topoisomerase solution. I tried increasing the centrifuge speed but the tubes tend to get clogged with the HMW DNA so nothing at all passes through. Size selection is a good idea too - normally I do a Blue Pippin 15 kb cutoff for a 20 kb library which collects everything over 15, but I could try defining the size range and see how that goes, thanks. Yes I know what you mean with the purifications - definitely an insane number! I've tried using Ampure beads on the BAC DNA but the beads just clump and they won't resuspend no matter what I do. thanks for your help!