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  • Long DNA isolation methods?

    Does anyone have a set of Minion-compatible long-read DNA fractionation methods (or links to same) that are willing to share? We have no problem getting DNA for the Minion up to 10KB, but we'd like to get up to 100KB.

    Thanks!

    Peter

  • #2
    Oxford Nanopore Technologies’ nanopore sequencing device, the MinION, holds the promise of sequencing ultra-long DNA fragments >100kb. An obstacle to realizing this promise is delivering ultra-long DNA molecules to the nanopores. We present our progress in developing cost-effective ways to overcome this obstacle and our resulting MinION data, including multiple reads >100kb.


    This preprint claims they can generate reads over 100kb. Please let us know if you can make it work. Good luck!

    Comment


    • #3
      what are you isolating from? I've had the most success getting large DNA out of environmental samples (bacteria and fungi, so difficult to burst cells) using the old school hot SDS and CTAB lysis followed by very gentle phenol chloroform extraction. Pipet gently-maybe even using wide bore tips. I haven't tried the minion yet, I was making fosmid libraries
      Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

      Comment


      • #4
        Hi,

        We are starting with bacteria, gram-negative rods. I suspect getting HMW DNA won't be a problem, but getting it sized correctly will be a challenge. The Covaris g-tubes only seem to 'accidentally' produce the 100Kbp+ long reads at a very low frequency, but are the only thing on the market I can find until you go up to expensive equipment like the BluePippen.

        Because we have a few (~reference) genomes that are somewhat closely related, I'm considering a restriction enzyme digest with rare-cutters to get extra long, easily repairable fragments (+1 for defined lengths!), and mixing them with the largest outputs I can get from a Covaris tube. We have an older Diagenode shearing instrument, but they just don't help people trying to develop new protocols... unless you buy a new instrument from them.

        We will likely need PFGE to confirm library sizes as a Agilent Tape Station only claims accuracy to 60KB.

        BUT... I was hoping someone could either share a working protocol or tell me what NOT to try. We've been with the ONP MinION access program, and while the computational people are doing great, we are hoping to improve the starting preps. Down the road we are going to be working on wheat and other crops.

        Thanks,

        Pete
        Last edited by hoytpr; 05-06-2016, 09:12 AM. Reason: added reference genomes

        Comment


        • #5
          Oh yeah, forgot to mention. I ran PFGE and cut out the desired size. Like I said, old school
          Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

          Comment


          • #6
            You can't get much more "old school" than me. And proud of it!

            I see this as the gold standard right now for generating ONP long-reads:

            http://biorxiv.org/content/early/2015/05/20/019281 by John Urban.

            If we can get this kind of distribution I'd be satisfied. But we still want to see if we can get longer reads... someday we are bound to need them.

            Comment


            • #7
              Originally posted by hoytpr View Post
              Hi,

              We are starting with bacteria, gram-negative rods. I suspect getting HMW DNA won't be a problem, but getting it sized correctly will be a challenge. The Covaris g-tubes only seem to 'accidentally' produce the 100Kbp+ long reads at a very low frequency, but are the only thing on the market I can find until you go up to expensive equipment like the BluePippen.

              Because we have a few (~reference) genomes that are somewhat closely related, I'm considering a restriction enzyme digest with rare-cutters to get extra long, easily repairable fragments (+1 for defined lengths!), and mixing them with the largest outputs I can get from a Covaris tube. We have an older Diagenode shearing instrument, but they just don't help people trying to develop new protocols... unless you buy a new instrument from them.

              We will likely need PFGE to confirm library sizes as a Agilent Tape Station only claims accuracy to 60KB.

              BUT... I was hoping someone could either share a working protocol or tell me what NOT to try. We've been with the ONP MinION access program, and while the computational people are doing great, we are hoping to improve the starting preps. Down the road we are going to be working on wheat and other crops.

              Thanks,

              Pete
              Hi everyone,

              I'm going to start a project to sequence the genome of gram-positive actinobacteria with long-reads in a MinIon instrument.
              I would like to know if you finally get a good protocol or kit for genome DNA isolation that could share, together with tips, for this purpose.

              Thank you in advance

              Comment


              • #8
                Originally posted by mrsrankind1990
                Hi. Can I contact you personally and discuss some details regarding your project?
                Hi mrsrankind1990,

                Yes, you can contact me personally. I'm pretty new in this forum, so I don't know how to send you a private message. I will try it by adding you to my contact list...

                Comment


                • #9
                  I have had a good experience with Circulomics Nanobind kits (I extracted DNA from brain, but there is a similar kit for cells and maybe also bacteria) and with very gentle phenol chloroform extraction (mixing the phases very slowly, e.g. 10 rpm, on a rotating wheel until fully emulsified gave the best quality).

                  Comment


                  • #10
                    Instead of covaris G-tube use syringe with needle for framentation.

                    Unfortunately covaris G-tubes have too small opening for getting DNA bigger than <10kbp, and fast flow generates a lot of shotr fragments.
                    for hitting 20-49kbp fargments range I recommend using a 5 - 10 ml syringe with G21 or larger needle and do 10 or more passes (in ~2ml of working volume) .

                    Also you can use circulomix short fragment elimination kit after syringe fragmentation.

                    Comment


                    • #11
                      There are two new kits; one from Circulomics and one from ONT that will become available sometime this Spring (I'm guessing this March??) that will have commercially vetted kits for Ultra-Long Reads on ONT.

                      Circulomics: https://www.circulomics.com/store/Na...Kit-p221284078

                      Meanwhile, there's this: https://www.protocols.io/view/rocky-...ple-pr-7euhjew

                      And this: https://www.biorxiv.org/content/10.1...471v1.full.pdf
                      Last edited by cement_head; 02-27-2021, 08:05 AM. Reason: clarification

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