This is why I am here...

[hydroshear]

Hi,

I am an expert in DNA shearing using hydrodynamic force (and other methods, because I have compared such methods side-by-side and know which is better for which situation). Feel free to ask me questions if you are uncertain what to choose! I would also like to learn more about third-generation sequencers and all the issues related to the $1k genomes.

[malachig]

Ok, I'll bite. In your opinion which method is best (and why) for generating RNA-seq libraries? Same question for whole genome libraries. Did your side-by-side comparisons include an evaluation of the randomness of each approach. That is, do some methods produce greater bias in breakpoints during shearing? If so, can this be related to GC content, secondary structure, etc.? Did you evaluate any methods that did not utilize hydrodynamic forces (e.g. enzymatic approaches)?

[hydroshear]

In your opinion which method is best (and why) for generating RNA-seq libraries?

My opinion is based on the fact that HydroShear has been successfully used to fragment mRNA. I have not heard anything about successful use of any other fragmentation methods (including those which Covaris advertises) on mRNA, so it is difficult to compare. If you have such data, I would be glad to make a comparison, providing that you share the data with me.

Same question for whole genome libraries.

If you are cloning DNA fragments, hydrodynamic shearing is better because the fragment's ends are less damaged. Also, HydroShear is the only system which can predictably and reproducibly generate large (>5kB) DNA fragments. On the other hand, it was not useful (so far) for making fragments smaller than 0.8kB.

Did your side-by-side comparisons include an evaluation of the randomness of each approach. That is, do some methods produce greater bias in breakpoints during shearing? If so, can this be related to GC content, secondary structure, etc.?

These questions are too specific for general discussion. We can discuss these with you, but since we do not perform such evaluation in our lab, such discussion is going to be mostly theoretical (I am aware of the small number of recent publications trying to address the issue, but my opinion is that these publications were, unfortunately, quite one-sided). We have done comparison by some other parameters which are more frequently requested by our customers, namely: run-by-run reproducibility, tightness of the fragment size distribution, intactness of the fragment's ends, estimation of the overall degree of damages the resulting fragments suffered, time spent and price per sample prep, etc. By the way, how can you truly evaluate the randomness of a method of DNA fragmentation if you cannot estimate the level of bias associated with the sequencing methods? (cloning, "cloning", etc)

Did you evaluate any methods that did not utilize hydrodynamic forces (e.g. enzymatic approaches)?

We did not, but enzymatic methods are obviously more expensive and more structure-dependent. Again, no one yet (to my knowledge) has succeeded to automate these.

[Hamid]

the Covaris AFA technology is very capable of shearing total RNA, as well as mRNA. We have quite a few customers who are actively using our technology in RNA-seq library preparation for SOLiD, as as Illumina platforms.
I have attached a sample data kindly provided by a few of your customers.

Also please follow the link http://covarisinc.com/supported-protocols.html for our 2kb, 3kb, and 5kb DNA fragmentation protocols, as we as gel and BioAnalyzer data.

Thank you

Hamid

[Hamid]

the Covaris AFA technology is very capable of shearing total RNA, as well as mRNA. We have quite a few customers who are actively using our technology in RNA-seq library preparation for SOLiD, as as Illumina platforms.
I have attached a sample data kindly provided by a few of your customers.

Also please follow the link http://covarisinc.com/supported-protocols.html for our 2kb, 3kb, and 5kb DNA fragmentation protocols, as we as gel and BioAnalyzer data.

Thank you

Hamid

[kevotu]

Quick question about the hydroshear, did anyone did any research on the actual mechanism/physics behind hydrodynamic shearing. How is it being shear? Is the terminal velocity in the orifice determine the fragment size or is it the ratios between the tubing diameter and the orifice? How much pressure/force would it take for the initial strand to break off?
What other methods or technologies that out there that could potentially play a role in shearing?

Any insights on these questions would greatly appreciated.