Construction of high-quality sequencing libraries is pivotal to successful NGS, and DNA quality is one of the most critical aspects of library preparation. As this Nature Methods paper illustrates, DNA shearing involves appropriate and consistent fragment sizes for sensitive and accurate sequencing, and the fragments must be accurately analyzed prior to sequencing to measure molarity and distribution.
Overview: DNA Shearing Methods
There are 3 approaches to DNA shearing: physical (including sonication), enzymatic, or chemical. Acoustic sonication, the preferred method, uses ultrasound waves to break apart chemical bonds.
Sonication is ‘cleaner’ than the other shearing methods because it doesn’t use enzymes or chemicals that may be carried throughout the sequencing workflow, negatively impacting read quality. In particular, sonication is superior in generating evenly sized fragments than shearing with specific restriction endonucleases, because these enzymes target specific points along a DNA sequence without regard for the distance between those points.
Ultrasonicators shear DNA by applying bursts of ultrasound, which cleaves hydrogen bonds and causes single- and double-strand ruptures of the DNA helix. Using this method, you can shear DNA into fragments as small as 100 base pairs in length. Exposure of DNA to uniform bursts of ultrasound leads to unbiased shearing, resulting in high yields of evenly sized double-stranded DNA for library preparation.
Ultrasonicators, such as Diagenode’s Megaruptor®, Bioruptor®, and Bioruptor® Pico, disrupt DNA strands through cavitation. This process creates and subsequently destroys of air bubbles and results in small, evenly sized DNA fragments. Sheared DNA obtained from this process is then analyzed to ensure optimal fragment sizes upstream of library preparation.
Best DNA Shearing Practices
Just as accurate and reliable NGS depends on library construction from high quality DNA fragments, the sonication fragmentation process depends on high quality starting material. Your DNA should be freshly purified, free of DNases, and have an OD260/280 ratio between 1.8 and 2.0. You must also ensure that your DNA extraction protocol circumvents cross-contamination by RNA, other existing small nucleic acid species, proteins, and other cellular macromolecules. These tips and reminders will help to ensure high-quality DNA shearing for successful sequencing experiments.
- Select the appropriate shearing tubes – Your tubes must be compatible with your sonicator. For example, 0.5 mL microtubes are recommended for use with Diagenode’s Bioruptor. You must also ensure that the caps can be moved out of the way, so that they won’t be damaged by contact with the sonicator. Tube plastic “hardness” is also important, to avoid damage from heat or ultrasound waves.
- Measure the right sample volume and concentration – Lower volumes (<50 µl) and higher concentrations generally yield poorer results, because of alterations of ultrasonic wave distribution in sample fluid. We recommend using volumes of 100 µl, and a sample concentration between 1 and 20 ng/µl, with an ideal concentration of 10 ng/µl.
- Keeping cool – Ultrasound wave propagation always produces heat that carries the risk of denaturing your DNA. Always keep your samples cold and on ice until you are ready to use them. A cold room alone will not keep your samples sufficiently chilled. Sonication is more efficient at 4°C, partly because of water’s highest density at that temperature.
- Optimize cycle numbers – For certain sample types and concentrations, you must make minor adjustments in cycle number to get the best results. You should set up time dose-response experiments to determine your most appropriate cycle number. Starting material containing very large fragments may require a longer dose to ensure homogeneous shearing.
- Take your samples for a spin – Quickly spin your samples between cycles to collect the liquid at the bottom of the tube. This ensures more efficient sonication. Remember to keep your samples on ice!
Where to Find DNA Shearing Support
The technology supporting ultrasonication for DNA shearing is evolving rapidly, offering a wider range of options for your experiments. DNA length is no longer restricted to 20 kb or less, and Diagenode’s Megaruptor® 2 can remarkably prepare fragment libraries up to 75 kb. But, no matter what technology you use, good technique is critical! You can find out more about DNA shearing by looking at Diagenode’s protocol and products pages here.Image Credit: soychemist