Improve Your Germline Testing by Optimizing DNA Fragmentation

Debadeep (Deb) Bhattacharyya, MSc, PhD, is the senior global director of marketing at Covaris, where he applies his expertise and experience in critical focus areas, such as protein analysis, genomics, epigenetics, and next-generation sequencing. He has authored more than 50 peer-reviewed journal articles and led the launch of multiple cutting-edge technologies, including LC-MS based solutions.

What is germline testing, and why is DNA fragmentation a necessary step in this process?

Germline testing identifies genetic variations or mutations in an individual’s DNA that can be passed down to their offspring, unlike somatic mutations, which are not heritable. Germline testing has multiple applications, including for cancer detection, where it helps determine if a tumor is hereditary or acquired. Genetic analysis of blood samples or oral swabs can be used to assess the presence of specific DNA sequences related to known oncogenes.

After sequencing a DNA sample, the sequence is compared with known gene libraries to identify oncogenes. However, before sequencing, DNA samples first need to be fragmented to a specific length to ensure accuracy.

What are the key challenges of DNA fragmentation, especially for germline testing? How well do current methods address these challenges?

When fragmenting DNA for germline testing, you are not always working with tissue. You may be working with whole blood samples so you must lyse the cells, or with whole fluid where you have many contaminants. Thus, a critical challenge is extracting enough high-quality DNA that is free of contaminants.

For DNA fragmentation, labs primarily use sonication, enzymatic fragmentation, or mechanical shearing such as Covaris’ Adaptive Focused Acoustics® (AFA) technology. These last two methods are particularly favored for DNA short read sequences (150–500 base pairs).

Except for AFA, the remaining methods have bias and throughput issues: If your technology cleaves only G-C pairs and not A-T pairs, you bias fragmentation, making it difficult to achieve sequencing accuracy with downstream assays. Additionally, the more samples you have, the more scalability becomes a concern.

Unlike methods such as enzymatic fragmentation, AFA is a mechanical fragmentation process, so it is noninvasive, doesn’t exhibit bias in cleaving DNA fragments, and isn’t affected by sequence preferences, allowing for robust processing of various sample types. Finally, AFA can accommodate the processing of multiple samples simultaneously, enabling scalability.

How does AFA compare with other methods in terms of efficacy for DNA fragmentation and recovery?

By enabling control over how long you expose the sample, AFA allows you to optimize conditions for extracting the highest amount of DNA from your specific sample. For example, you can shorten exposure for liver cells compared to skin cells, which improves yield of high-quality DNA from the more fragile liver tissue. This fine control is not available with methods like enzymatic fragmentation.  

Along with DNA, AFA enables extraction of RNA, which is often destroyed by methods like enzymatic fragmentation, but can offer an extremely valuable, desirable, and complementary confirmation of the presence of oncogenes. Importantly, AFA works with fresh frozen and soft tumor tissue, FFPE samples, fibers, cell lysates, and organoids.

As germline testing becomes commonplace, clinical labs need to identify how they will produce high-quality data for patient care. Opting for cost-effective, multipurpose, unbiased, and customizable technologies like AFA for DNA and RNA extraction, as well as DNA shearing, helps labs produce scalable and reliable results.