What is fragmentation?
Fragmentation is a key step of an NGS library preparation workflow. During the fragmentation process nucleic acid molecules are randomly sheared or fragmented to generate a pool of fragments that are of a similar size.
Fragmentation is usually included in library prep for short read sequencing. The length of your fragments depends on the sequencing instrument and samples being used. Not all NGS workflows will require fragmentation, especially if the nucleic acid molecules being prepared for sequencing are already highly degraded (e.g., cfDNA, DNA extracted from FFPE tissue, or damaged RNA).
There are two types of DNA fragmentation, mechanical and enzymatic, and depending on your research application, the choice between the two could play a major role in determining the quality of your NGS library.
What is the difference between mechanical and enzymatic fragmentation?
Mechanical fragmentation, also called mechanical shearing, involves the use of a physical force to break up DNA molecules. This includes things like acoustic shearing, sonication, and nebulization. Enzymatic fragmentation involves the use of enzymes to fragment DNA. Examples of enzymatic approaches include the use of nicking enzymes, restriction enzymes, or transposases.
The choice between mechanical vs enzymatic fragmentation should be considered carefully as the fragmentation process, if completed incorrectly, can result in over fragmentation or over digestion, resulting in DNA molecules that are too short and unsuitable for downstream library preparation. The following section discusses important factors to consider when choosing between fragmentation approaches.
How to decide between mechanical and enzymatic fragmentation
The best method of fragmentation for your NGS workflow will depend on several factors:
- Concentration of sample input: It's important to keep in mind that the use of mechanical fragmentation may result in material loss due to the extra handling/transferring of samples to perform the mechanical shearing process, while enzymatic fragmentation minimizes extra sample handling as adapter ligation can be performed in the same tube as the fragmentation occurred. Therefore, if the amount of sample being used is limited, enzymatic fragmentation is recommended as this approach can reduce the amount of sample lost in between library prep steps.
- Number of samples: Mechanical shearing approaches can be limited in the number of samples that can be processed in parallel, making enzymatic fragmentation an attractive choice if you have a large number of samples needing library preparation at the same time. Additionally, enzymatic fragmentation can be incorporated into automated library prep workflows easily, while mechanical fragmentation may require samples to be fragmented before being placed onto an automation platform.
- Enzymatic sequence bias: One potential drawback of enzymatic fragmentation is that the enzymes used to breakup DNA fragments could exhibit sequence bias, generating non-random fragmented molecules. Randomization during fragmentation is important to ensure that the resulting sequencing library is truly representative of the starting sample. Mechanical fragmentation does not come with the risk of sequence bias as all samples are run with the same settings, regardless of GC content. There are DNA library prep kits that have been shown to generate high-quality NGS libraries across various ranges of DNA GC content. Read more about these kits here.
- Frequency of library preparation workflows: Most mechanical shearing approaches require a machine. Therefore, if NGS library prep is not a common occurrence in your lab, it may not make financial sense to purchase an instrument that will rarely be used. Hydrodynamic shearing (using a syringe or centrifuge to fragment DNA) is an affordable mechanical fragmentation option, however the amount of input DNA required for this approach is high and the number of samples that can be fragmented in parallel is limited. Enzymatic fragmentation does not require special instruments outside of what would be required for library preparation.
DNA fragmentation tips and tricks
- Clearly define your research objectives. As mentioned in the previous section, the decision between mechanical vs enzymatic fragmentation depends on various factors, some of which may be out of your control –i.e., concentration of sample input. However other factors that you will have more control over, such as which sequencing platform and read length, should be considered so that your research goals will be achieved.
- Avoid over doing it. In other words, carefully read the protocol you are following or the documentation for your mechanical shearing instrument. For enzymatic fragmentation, over digestion can occur if samples and enzymes are mixed and left at room temperature. If mechanical shearing is the approach you’ve chosen, then be sure to check the fragmentation setting recommendations for your instrument.
- Practice makes perfect. It is also wise to test the approach you’ve chosen with a subset of replaceable samples. This allows you to optimize your fragmentation approach and determine where adjustments can be made without losing precious samples.
Need more information about fragmentation?
When navigating the landscape of NGS library preparation there are many factors to consider such as sample input (type, quality, etc.), different sequencing chemistries, library normalization approaches, and more. If you want a comprehensive guide to help you get started with your library prep workflow, check out the Next generation sequencing guide here.
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