High-Throughput DNA Shearing with FastPrep

The Importance of DNA Shearing

Next generation sequencing (NGS) has revolutionized life sciences research. With NGS, we can explore genomes deeper, genetically characterize microbial communities, and better understand complex diseases.

To generate reliable NGS data, you need to construct high-quality sequencing libraries, which largely depends on DNA quality. Libraries are built using DNA fragments generated via enzymatic or mechanical shearing of DNA from your samples.

Optimal DNA shearing methods deliver:

1. Appropriately and consistently sized DNA fragments (depending on sequencing platform)
2. Unbiased shearing where fragmentation is not preferential towards certain sequences or GC content
3. Pure DNA samples—mitigating cross-contamination between samples
4. High DNA retention where the shearing method avoids sample loss

A Validated DNA Shearing Protocol

The FastPrep-96 instrument mechanically shears DNA, generating fragments using mechanical force rather than enzymes, such as nucleases, to cleave DNA. With the FastPrep-96 instrument, you can confidently fragment 100-1000 ng of genomic DNA (gDNA) in high-throughput—shearing up to 576 samples within minutes. Here’s how:

1. Adjust the volume of gDNA to 50 μl with nuclease-free water and transfer to a 96-well plate.
2. Seal the 96-well plate with an Adhesive PCR Plate Foil seal.
3. Load and secure the 96-well plate(s) onto the FastPrep-96™ system (you can fit up to six 96-well plates in the instrument.
4. Run the FastPrep-96™ system at the appropriate speed and time to fragment gDNA to the required size.
5. Analyze the fragmented DNA to assess fragment size (use the Agilent Bioanalyzer 2100 or Agilent Femtopulse)

From here, you can prepare your newly fragmented gDNA for sequencing by using a Ligation Sequencing Kit.

Selecting the Appropriate DNA Shearing Speed and Duration

Read lengths vary based on fragmentation time and speed. Adjust the fragmentation time at 1800 rpm to generate uniform fragment lengths, rather than changing shearing speed.

For shorter read lengths, you’ll need to increase the shearing time, or consider an alternative DNA shearing method.

Alternative DNA Shearing Methods

There are several alternatives to using FastPrep systems for DNA shearing.

Acoustic cavitation is commonly used to fragment DNA, which can be accomplished by sonication or Adaptive Focused Acoustic (AFA) shearing. You’ll minimize DNA loss with probe-sonicators, but the method may require time-consuming calibration to avoid fragment size variability. AFA shearing has many advantages, but it is costly.

You can also enzymatically shear DNA by using a combination of a non-specific nuclease and T7 Endonuclease I to generate fragments in a non-biased manner, but cross-contamination and sample loss can be an issue.

You should choose your DNA shearing method based on DNA fragment size requirements, abundance of DNA, number of samples, and budget.

Not Just Mechanical Shearing of DNA

In addition to DNA shearing for library preparation, you can use FastPrep-96, or any other FastPrep Instrument, in your sample preparation workflow for NGS—transforming multiple steps in your process from time-consuming to high-throughput. Our FastPrep systems are known for their high performance in cell lysis and bead beating, with the ability to reliably homogenize almost any sample when using the right lysing matrix.