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Old 10-20-2009, 06:47 AM   #1
pmiguel
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Default New fragmentation methods

Physical shearing methods for double stranded DNA include sonication, nebulization and hydroshearing. We use all three. They work well for constructing a handful of libraries. But without specialized equipment/automation, constructing larger numbers of libraries really starts to become a bottleneck.

Just in the last week I have seen advertisements for two new fragmentation methods. One, from New England Biolabs (NEB), "dsDNA Fragmentase", and another from Epicentre Biotechnologies, "Nextera".

NEB's ds Fragmentase uses a mutant Vibrio vulnificus nuclease and a mutant T7 endonuclease (both fused with maltose binding protein for reasons unclear to me) and another from Epicentre Biotechnologies deploys an engineered transposase

Quote:
one randomly generates nicks on dsDNA and the other recognizes the nicked site and cuts the opposite DNA strand across from the nick, producing dsDNA breaks. The resulting DNA fragments contain short overhangs, 5-phosphates, and 3-hydroxyl groups.
More information available at the NEB web site:

http://www.neb.com/nebecomm/products/productM0348.asp

An aside here: As scientists I think we should demand that commercial entities wanting to sell us products reveal the compositions of those products. Lamentably this has become increasingly rare. But NEB displays a level of transparency in this regard that should be required of all companies wanting to transact with researchers using public funding. Just wanted to mention that. I have no connection with either NEB or Epicentre, other than I buy their products.

Epicenter's Nextera uses their "Transposome" technology to randomly bomb DNA. The ends of their engineered transposon apparently are not connected, so each insertion generates two ends (a break), each terminated with an appropriate adaptor. The trick in next gen library construction is always getting adaptor "A" on one end and "B" on the other. Since the "A" and "B" ends of the Transposome end up terminating different molecule, we would still expect half the fragments generated to be "A-A" and "B-B". But that is no worse than ligation methods deliver, and it cuts out the need for a ligation step. More information here:

http://www.epibio.com/nextera/nextera_tech_overview.asp

Just to be clear about the benefits, physical fragmentation methods generally require substantial "hands on" time for every library being constructed. That is, you take the DNA sample, you load it into the device (nebulization unit, sonicator, or hydroshear), perform the procedure, then unload. Even ignoring issues like the potential for cross-contamination and maintenance costs for these devices, scaling is an issue. Making 10 libraries is do-able. But 96?

So the allure of non-mechanical methods is obvious--you could probably set up either of these in a 96 well plate and process using multichannel pipettors, or even use laboratory robotics. But let us not forget why we flocked to mechanical methods in the first place: enzymes never seem to be as random as the mechanical methods. Being derived from living systems they come with their own agenda, so to speak. And that agenda usually results in bias. My advice would be to pay attention to the tests both companies did on differential depth of coverage for libraries generated using their methodologies.

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Old 10-20-2009, 03:59 PM   #2
Hamid
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Hi Phillip,

Thank you for the information on the NEB fragmentase, and the Epicenter's Nextera.
I just wanted to mention that aside from the Nebulization, hydroshearing, enzymatic digestion, and sonication, there is a 4th choice, and that is the Covaris Adaptive Focused Acoustics technology for DNA shearing. It is a highly efficient non-contact, closed vessel, isothermal method of shearing DNA. We have protocols and validated settings to generate tight distribution of fragments with average peaks ranging from 100bp-5kb. Our E210 instrument is capable of processing 96 samples in batch mode. It is very much in use and part of the sample processing for library preparation at Broad, JGI, and Sanger institute just to name a few. Our technology is used for library preparation on the Life Technology's SOLiD, Roche 454, and Illumina GAs,
Our protocols are located at http://covarisinc.com/supported-protocols.html. You can also take a look at some of the publications from Broad, JGI, and Sanger located at http://covarisinc.com/publications.html#app_a in which they discuss the use of the Covaris AFA for DNA shearing.
I will be more than happy to discuss our technology further, and answer any questions you might have.

Hamid
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Old 10-26-2009, 05:30 PM   #3
yuyak
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Thanks Phillip for the information. I saw the ad for the NEB fragmentase and I could not figure out what exactly the use for it was. What's the advantage of using this over something like DNaseI or MNase?

Nextera looks really promising, at least conceptually. I look forward to hearing more about it.
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Old 10-27-2009, 04:23 AM   #4
pmiguel
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Quote:
Originally Posted by yuyak View Post
Thanks Phillip for the information. I saw the ad for the NEB fragmentase and I could not figure out what exactly the use for it was. What's the advantage of using this over something like DNaseI or MNase?
I could only speculate. Possibly NEB searched for an enzyme lacking biases that DNaseI or MNase might have. Past that, the advantage of using a kit specifically designed for your application is that the designers of that kit have probably already dealt with at least the first set of technique hurdles one would run into designing a protocol on ones own. But, obviously, if you already have a methodology working to your satisfaction using another enzyme, then fragmentase may not be worth looking into.

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Old 10-27-2009, 04:47 AM   #5
pmiguel
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Quote:
Originally Posted by hamid View Post
Hi Phillip,

I just wanted to mention that aside from the Nebulization, hydroshearing, enzymatic digestion, and sonication, there is a 4th choice, and that is the Covaris Adaptive Focused Acoustics technology for DNA shearing.
Hamid
Hi Hamid,
Yes, an investigator here at Purdue has one of these instruments. However, I would class this method as "sonication" -- although, yes, it does directly address the major limitation of most physical shearing methods. That is, fragmenting larger numbers of samples.

Has anyone looked at what % of ends produced by sonication are enzymatically repairable such that they can be ligated? I posted about the study I'm aware of:

http://seqanswers.com/forums/showthread.php?t=2759

There I raised the possibility was raised that some of the ends produced by sonication might not be repairable. New England Biolabs has an assay they perform for restriction enzymes which states "blah% of fragments produced by this enzyme could be religated". I wonder what a similar assay would say about DNA fragmented by various mechanical means and then end-repaired?

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Old 10-27-2009, 09:52 AM   #6
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I have been involved in the development of the Epicentre protocol (although I hate the name they came up with)--we are still looking at insertion bias. I'm looking forward to switching over for 454 sequencing; it is so much simpler than the standard library prep methods. I'm sequencing mostly very small genomes or constructs, so some bias is acceptable to me vs. the ability to make many libraries in parallel.
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Old 12-25-2009, 10:52 PM   #7
hanjaylee
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Hi Phillip,
i find you always posting very useful and constructive messages. i really appreciate that.
i want to ask if i want to construct a human genomic library with inserts ranging from 10kb to 15kb. which method will you recommend? thanks a lot.
hanajay
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Old 12-26-2009, 10:03 AM   #8
pmiguel
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Quote:
Originally Posted by hanjaylee View Post
Hi Phillip,
i find you always posting very useful and constructive messages. i really appreciate that.
i want to ask if i want to construct a human genomic library with inserts ranging from 10kb to 15kb. which method will you recommend? thanks a lot.
hanajay
Thanks Hanajay,
I put some effort into this for a number of reasons. Some include:

(1)"Paying Forward" the help I got from various forums over the years: bionet.autoseq, ABRF and maybe the most unsung of benefactors, Bruce Roe's web site, http://www.genome.ou.edu/

(2)Trying to help the seqanswers forum to foster an environment were next gen sequencing can be discussed at a detailed level. Especially if it helps to alleviate the shroud of pseudo-secrecy that contaminates scientific protocols. I think this bizarre posture that most science reagent/equipment companies embrace is counter-productive because it cuts them off from an extremely valuable source of information--their own customers.

Anyway, as far as the library you want to construct, we have never done large insert mate pair libraries for 454 (or any other next gen platform). But the Roche protocol looks pretty good to me. Have you given that a try?

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Old 12-26-2009, 05:04 PM   #9
hanjaylee
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Quote:
Originally Posted by pmiguel View Post
Thanks Hanajay,
I put some effort into this for a number of reasons. Some include:

(1)"Paying Forward" the help I got from various forums over the years: bionet.autoseq, ABRF and maybe the most unsung of benefactors, Bruce Roe's web site, http://www.genome.ou.edu/

(2)Trying to help the seqanswers forum to foster an environment were next gen sequencing can be discussed at a detailed level. Especially if it helps to alleviate the shroud of pseudo-secrecy that contaminates scientific protocols. I think this bizarre posture that most science reagent/equipment companies embrace is counter-productive because it cuts them off from an extremely valuable source of information--their own customers.

Anyway, as far as the library you want to construct, we have never done large insert mate pair libraries for 454 (or any other next gen platform). But the Roche protocol looks pretty good to me. Have you given that a try?

--
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that is nice and meaningful! i will go check the Roche protocol
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Old 02-19-2010, 11:41 AM   #10
angelawu
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Quote:
Originally Posted by hanjaylee View Post
Hi Phillip,
i find you always posting very useful and constructive messages. i really appreciate that.
i want to ask if i want to construct a human genomic library with inserts ranging from 10kb to 15kb. which method will you recommend? thanks a lot.
hanajay
With regard to the large mate pair libraries, you might find this page helpful, particularly in the "More on Fragmentation" section.

http://genomecenter.ucdavis.edu/dna_...equencing.html

They claim success using the new NEB fragmentase enzyme, so it might be worth a try.

Good luck!!!
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Old 03-09-2010, 12:22 AM   #11
Gillow
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Has anybody obtained fragments under 1Kb (with most in the 500 Mb to 800 Mb range) consistently in any fragmentation method.
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