Covaris, a necessity?

[cli]

Don't get me wrong, I know Covaris is the best for its job. But if you have a small budget and no genomic center around, can you get around it by using enzymes, even just DNase I. What is the pros and cons of doing that? Thanks.

[Heisman]

You can probably get around it with a bioruptor if you're willing to spend a bit of time testing different settings.

[Rocketknight]

You can do the job with enzymes (NEB fragmentase or something) at the cost of a broader size distribution. You'd need to do a gel-cut size selection most likely, and you'll need some extra initial sample to compensate for the fact that a lot of the sheared DNA will probably fall outside your desired size range. Nonetheless, if you can put up with that your library quality should still be fine.

[TonyBrooks]

Have you looked into Nextera library prep?
It's much cheaper through Illumina than it was through EpiCentre and it works well for us. Still not as cheap as standard preps, but it saves on having to spend on a Covaris - not cost effective if your throughput is low....and it's much, much quicker.

[cli]

Thank you all for the reply.
TonyBrooks, how is the size range of Nextera prep? Do you need to do a gel-cut size selection afterward? Can you produce library with different size? e.g., 200bp, 400bp. It make sense that Nextera combine the fragmentation and adapter adding in the same step, which may increase the yield of the library. Have you seen that in your experience? Finally, how much the kit cost? I can't find out the price even after I register on Illumina webpage. Sorry for so many questions. I appreciate your advice.

[TonyBrooks]

You do get a large size distribution (250-1000bp) but this is sufficient for us. Most distributions are tighter than that when looking at the sequencing. You could always augment the reaction with a gel-cut as in regular sample prep. You might be able to add more starting material to compensate for the loss of material from the gel excision.

[cli]

Tony, thanks a lot for the information. I guess I have to give it a try and find out if it works for us.

[TonyBrooks]

I should also mention that Nextera isn't properly supported by Illumina for target enrichment - only for standard libraries.

[cli]

Can you elaborate it a bit more about why Nextera can't be used for target enrichment? If it can be used for standard Illumina libraries, the only things needed are blocking oligos for adapter sequences if I am correct.

[TonyBrooks]

I'm not saying you can't do it, just that it's not officially supported by Illumina (certainly for TruSeq enrichment, not sure about Agilent and Nimblegen).
With the correct blocking sequences, you should be able to cobble something together. You also probably wouldn't need to gel-cut as target enrichment seems to create tighter libraries.

[Hamid]

Hi Cli,

I think the investment in sample preparation is sometimes overlooked in NGS which can lead to more work, sample loss, and adverse effects on the quality of data obtained.
While comparing other shearing methodologies it is important to consider the following:
1. time and work required to optimize the shearing process. Covaris does have validated optimized settings for all size ranges from 100bp-5kb. These settings are practically concentration independent.
2. sample loss. how precious are you samples. no direct contact with the sample, so no sample loss, and no worry for contamination.
3. flexibility in shearing size range.
4. reproducibility. The Covaris instruments are hard wire calibrated in the factory, so absolutely no calibration required. All Covaris protocols will generate the same size range of interest on any of the instruments in any lab.
5. Thermal damage/sequence bias. isothermal processing achieved with the Covaris, so no worry for thermal damage, or thermal biased fragmentation.
6. Size selection prior to library preparation. With Covaris shearing the fragment distribution is tight enough so that you do not have to do size selection for quite a few of the library preparation protocols.


If you do have budgetary concerns, there are two options from Corvaris:
1. Use the Covaris shearing service.
2. wait for the upcoming release of the Covaris M220 which is a low cost shearing instrument from Covaris specifically designed for DNA shearing for low throughput labs.

Thank you