Are you working with DNA, RNA, etc? The problem with not mechanically shearing your say DNA with a covaris or even bioruptor is loss of DNA from a broad range fragmentation and increased contamination potential when you size select on a gel. If you have limited amount of precious sample I would recommend mechanical shearing. On the other hand from what I hear Nextera preps are faster.

thanks. the last set of samples were made with TruSeq, I made some with Nextera which will be sequenced soon, so we can do a direct comparison. It is faster, and not having to find a Covaris is good too.

Mechanical shearers are ridiculously pricey jewelry cleaners. Please keep me updated on the end result comparison, especially in regards to potential sequence bias. Slightly off topic, do you use pippin prep for size selection?

What's the difference in pricing between the kits?

the cheapest way to go for Illumina library prep kits are the NEB modules. But Id prefer to use the Nextera if it give similar results because doing the Corvaris shearing is at least $5 a sample, if you can find someone to do it. And nextera keeps the reactions in the same tubes. sending the samples out to get sheared then returned isnt something id prefer to do. otherwise both kits will be around 75 a sample..which is cheaper than the 300-500 youll pay to have it done at a core lab - and all you need is a thermal cycler and a gel box.

Carlos, Thank you for the thoughts. Makes sense to compare to the core lab, and when you put it that way I like your approach. I agree that sending the samples to be sheered would introduce an added label of complexity to the process and increase sources of errors.

I've found the NEB kit to be a bit confusing when trying match it to protocols on their website. For instance, the NEBNext #E6000 has blunt-ending reagents that are in different concentrations from the NEB Quick Blunting Enzyme Mix. This is a problem that can only be resolved by customer service because the NEB online protocols are only for Blunt Enzyme Mix (which is intended for cloning, not library preps which involves shearing) and not the separate tubes of kinase, polymerase and dNTP found in the E6000.

Here's the difference for blunt end repair.

The concentration of the Quick Blunting Enzyme Mix is:

0.05U/ul of T4 DNA Polymerase

1U/ul of T4 PNK.

using 1mM dNTP

The concentration of the enzymes in E6000:

10U/ul of T4 PNK

3U/ul T4 DNA Polymerase

10mM dNTP

You will have to dilute the reagents in E6000 down to the concentration of the blunting enzyme mix. You could, for instance, make up 30ul with 3ul of the T4 PNK and 0.5ul of the polymerase and use 1ul. Ideally the remaining volume should be brought up with something that has 50% glycerol or TE. You can make up a smaller volume if you have pipets that will accurately dispense the small volumes. The dNTPs can be diluted with water.

Here is a good paper that addresses the issue

http://www.ncbi.nlm.nih.gov/pubmed/21948828

Hi Carlos,

Thanks for alll he information provided

so based on your results, how did the end comparison between the Nextera and TrueSeq? on aspects of the potential sequence bias