DNA shearing

[suludana]

Hi,
these days I'm hearing a lot of polemic around DNA shearing in the first step of sample preparation protocol from Illumina.
Someone is using Covaris S2 or other kind of instrument instead of Nebulization?
What opinions and experiences you can share?
Thanks

[swbarnes2]

The Corvaris is pretty universally seen as the best way to shear. The size distribution is pretty tight.

Part of the reason that the Broad can do a no-PCR sample prep is because they don't do a gel extraction, and the reason they don't have to do one is that they trust their Corvaris to do a good enough job shearing to the size they want.

We wish we had one

[marklar]

bioruptor

we have been using the bioruptor for shearing LRpcr products as well as genomic DNA which is then sequenced on both 454 and Illumina platforms.

we decided to go with the bioruptor because it is wayyyyy cheaper and also because we can shear 12 samples simultaneously...though there is now an upgrade where you can do 24 at a time. on the covaris you can only shear one sample at a time.
So...the price and the number of samples you'll be working with should be used to make a determination.
Although the bioruptor gives a broad range of fragments, in comparison with the covaris we saw that it was very similar. We are able to typically shear down to between 150 and 300 to 400 bps. We also don't do gel cuts.

Nebulization, on the other hand, gives much broader size ranges and is a pain to work with.

[ScottC]

Well, you can also buy a Covaris unit that will process multiple samples, but it's even more expensive .

http://www.covarisinc.com/e_series.htm

The 'E' series processes 96 samples, the 'S' series processes single samples.

[Hamid]

Apologies for the late reply. I just joined this forum.
In response to swbarnes2: I am an Applications Scientist for Covaris, and I am located in the Orange County area, and if interested I can provide a demo of our technology using your samples to show how easy, and reproducible our technology is for shearing DNA to your specific size ranges.

[chen]

Hi everyone,
Here I have some questions with the Covaris.
The problems of the stability of Covaris really bother me during the second fragement when I am preparing 2-5Kb samples for MATE PAIR library sequencing. The parameters worked out in last experiment DO NOT WORK next time. Could anyone give me some advice on that?

Thanks,
chen

[Hamid]

Hi Chen,

Only just this week Covaris introduced protocols and consumables specifically designed for shearing of DNA fragments to 1.5kb(http://www.covarisinc.com/pdf/pn_400068.pdf), and to 3kb(http://www.covarisinc.com/pdf/pn_400065.pdf).
These protocols have been tested in-house and at customers sites for reproducibility, and tight shearing distribution.

[chen]

Thank you, hamid.

In our lab, usually we use Nebulizer(the FIRST fragment) for shearing of DNA fragments to 2-5Kb, and Covaris for fragmenting the Circularized DNA(the SECOND fragment) to about 600bp, but it came to be either smaller or larger. Any suggestions?

[Hamid]

Hi Chen,

Here are some questions that will help me diagnose what you are seeing:
1. What buffer are you using for shearing?
2. Which tubes are you using for the shearing?
3. What volume and what concentration of DNA are you shearing?
4. What is the serial number of the Covaris S2 instrument you have?
5. What settings and temperature are you using for the DNA shearing?

Thank you.

hamid

[chen]

I used glass tube with 3mg beads in it, and putting the whole mix into 300ul with TE. The concentration of DNA would be <5ug.

I had tried to lower the beads, and that worked better.

Thanks,
chen

[Hamid]

Hi Chen,

the problem seems to be that you are not using our validated protocols for shearing DNA. Where did you obtain the protocol you are using?

None of our shearing protocols for shearing DNA less than 3kb require the use of glass beads.
Please follow the shearing protocol at http://www.covarisinc.com/pdf/pn_400056.pdf .
Our protocols for shearing up to 5ug of DNA in a volume of 120ul using our Microtubes with the AFA fiber (part number 520045 or 520052) can be found in that pdf.

Also please provide the answers to questions 4 and 5.

Thank you

Hamid

[polivares]

Hi,
I would like to know the fragmentation size spectrum of the covaris s2. Some resources state that the minimum size is around 600 bp but others state that it is 60 bp. Besides, does reproducibility varies along fragment size?
Thank you

[jorgemon]

Hi polivares.
This link may help to solve your question...

http://www.covarisinc.com/supported-protocols.html

One of this protocols (for fragments <1.5kb posted by hamid before) said that you can get fragments between 100 and 1500bp.
We have done test with covaris S2 and we got reproducibility results in same samples (for sizes around 200bp).
Best regards.

[CompBio]

Since we are on the subject, could someone instruct me, a non-biologist? I've read a bit on the protocols used for RNA-Seq. I'm nominally familiar with RT-PCR and how different primers ought to work, and I've read about techniques such as nebulization for obtaining a consistent cDNA library size.

However, I don't understand how cDNA fragments 100bp to 2000bp long become sequenced reads of 40-50bp. Do you fragment the cDNA again? Is the sequencing done on these long fragments, but only reporting the first 50 calls?

If there's a publication on this subject (without too much biochemistry) I'd like to see it. Ultimately I'm trying to determine whether we can assume the cDNA fragments are uniformly distributed--both within a given mRNA transcript and across all transcripts--before sequencing even begins.

Many thanks!

[kmcarr]

The Illumina (née Solexa) sequencing method incorporates one nucleotide per strand at a time, detects which base was incorporated and then incorporates the next base, and so on and so on. The read length is determined by how many of these incorporation/detection cycles are performed. The template DNA fragment is longer than the read length but since a only finite number of cycles are being performed it does not matter how long the template is.