High Quality NGS DNA Library Construction by DNA Fragmentation with Covaris Ultra-sonication – When Size Really Matters!
The generation of high quality DNA libraries is critical for achieving adequate DNA sequencing depth and rate of coverage. Preparing sequencing libraries containing DNA molecules with a desired average length involves fragmentation using either enzymatic digestion, hydrodynamic shearing, or sonication.
Enzymatic digestion can be unsuitable in certain applications given its sequence-specific cleavage and biased susceptibility for specific chromatin regions. While some size selectivity can be achieved with hydrodynamic shearing, gaps in fragment coverage due to shearing biases have been observed.
Nebulization, which forces DNA through a small hole in a nebulizer unit, often requires an additional purification step to achieve the desired narrow fragment size range, which can lead to sample loss. Both probe and bath sonicators are notoriously unpredictable and can expose the DNA sample to high heat.
Sonication using a probe sonicator involving the direct contact with the sample can be problematic for small volumes as well as cause sample cross-contamination.
The ICBR utilizes the Covaris Adaptive Focused Acoustics™ (AFA) ultra-sonication technology for DNA fragmentation during library preparation.
AFA is considered the “Gold Standard” due to its technological advantages over other sample prep methods, such as sonication and nebulization.
Key advantages of AFA technology include reproducibility, versatility (DNA output across a wide size range), uniformity in fragment size distribution and isothermal, non-contact methodology.
The ICBR offers two Covaris instruments for self-use by UF researchers in preparing DNA libraries. For single-sample applications the S220 Focused-ultrasonicator is computer controlled to incorporate preset protocols for DNA shearing to specific fragment lengths, and easy-to-use-tools for user-defined protocols.
The Covaris E220 Ultra-sonicator enables multi-sample, batch preparation, capable of processing a wide range of sample types and volumes.
In addition to DNA fragmentation, the versatility of the E220 ultrasonicator makes it possible to bring the advantages of AFA to numerous biological and chemical applications including chromatin shearing, tissue homogenization, cell lysis, compound dissolution, and particle micronization.
If you are interested in learning more about using Ultrasonication for your library preparations or any other applications please contact Dr. David Moraga or Dr. Savita Shanker in the ICBR.