Next-generation sequencing (NGS) really has taken the world by storm! In NGS, millions of short ‘read’s are sequenced in a short space of time, leaving you with vast amounts of data to analyze! For all NGS platforms, the input sample (i.e. your cell free DNA) must be cleaved into short sections or fragments prior to sequencing. The fragment length depends largely on the particular sequencing platform used. Although it might seem as though everyone is sequencing genomes nowadays, it is still not a trivial undertaking with many important factors to consider. One of the biggest factors to consider when planning a NGS project is
DNA size selection (i.e., the size of your DNA fragments) and the method you will use to achieve this.
Fragmentation (or shearing) of DNA it is not as simple as it might sound and NGS quickly reveals the biases inherent in one protocol versus another. Choosing the shearing method depends on a few factors such as DNA size selection and how much DNA is available, as well as your budget!
Let’s have a quick look at main methods for DNA shearing:
1) Enzymatic Shearing
It might be obvious that this method involves digestion of
cell free DNA (
cfDNA) with some kind of enzyme, however the choice of enzyme is very important. Enzymatic shearing yields fragments through simultaneous digestion of both DNA strands, or by generation of nicks on each strand of dsDNA to produce dsDNA breaks. Enzymes which simultaneously cleave both DNA strands (e.g. restriction endonucleases) often exhibit sequence preference, thereby introducing bias. Therefore, a combination of non-specific nuclease and T7 Endonuclease I is often used to yield fragments in a non-biased manner. The nuclease nicks the DNA and the endonuclease cleaves the nicked DNA.
An important drawback with enzymatic shearing is the potential risk of bias. Since precise quantification is often a goal in NGS projects, physical fragmentation is often the preferred method for shearing. Fortunately, recent years have seen the emergence of a number of commercial enzymatic fragmentation kits, which claim to circumvent many of the issues associated with enzymatic shearing.
2) Nebulization
Nebulization, generating a heterogenous mixture of DNA fragments of 100-1200 bp in length, was popular in earlier sequencing projects. This works by forcing cfDNA through a small hole in a nebulizer unit, resulting in the formation of a fine mist of DNA fragments that is collected. DNA fragment size is determined by factors such as the gas pressure, the speed at which the DNA travels through the nebulizer, and the temperature. This is a reproducible technique that shows little or no sequence bias.
However, nebulization requires a large amount of DNA input. Typically 1 to 5 g are used with about half of the DNA being atomized and only 10 % of DNA being the right size for analysis. In other words, while nebulization offers the benefit of shearing DNA in a non-biased manner, it is an inefficient process, and is unsuitable in experiments involving many samples because of the very large amounts of input DNA needed.
3) Sonication
Sonication has been widely used for
DNA shearing. This technique uses acoustic cavitation to fragment DNA. Most probe-sonicators can be quite variable and require careful calibration to achieve the correct size-distribution. A potential drawback is that it can take 15 to 30 minutes to process a sample. Sonication generates fragments of about 700 bp in length, and very little DNA is lost making it a very attractive method.
4) Covaris shearing
One method that has been adopted in almost all high-throughput laboratories is
Adaptive Focused Acoustic shearing (AFA) which features in the
range of instruments from Covaris Inc. Acoustic energy is focused into a small glass vial containing the sample which induces cavitation of the aqueous sample. Cavitation is the continuous creation and collapse of micro-bubbles in the sample. This creates localized forces that fragment the DNA quickly and reproducibly.
Like nebulization and sonication, there appears to be little or no genome bias with this method, but because the size-range is narrower than nebulization or sonication (and there is no sample loss with Covaris shearing), much more DNA is available for library preparation. This makes Covaris shearing the preferred method for most NGS users.
Which Method for DNA Shearing Should You Choose?
When we were looking for a system to robustly generate sheared DNA fragments we evaluated several methods and settled on
Covaris. It generates uniform fragments in reasonable time and its closed-tube mode of operation means there is virtually no risk of genomic DNA contamination. If you need to process hundreds of samples you could use the plate-based model, but it is an expensive tool for the lab. Ah, if only money were no object!
