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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!
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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. -
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!Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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