Optimizing Germline Testing: The Importance of High-Quality Sample Prep (ASHG 2023)

At the recent ASHG 2023 meeting, Eugenio Daviso Ph.D., VP of Solutions at Covaris, shared the importance of precise DNA fragmentation in next-generation sequencing (NGS) for germline testing. Daviso highlighted the use of Covaris’s Adaptive Focused Acoustics^® (AFA^®) technology, which revolutionizes sample preparation by reducing assay failures, ensuring better coverage and uniformity, while significantly cutting costs. The impact of this technology extends to workflow efficiencies, improved quality of results and enhanced lab economics, particularly in germline testing workflows.

Introduction

Germline testing is crucial for assessing the risk of hereditary cancer and rare diseases. However, traditional enzyme fragmentation during NGS library prep presents significant challenges to ensuring trustworthy results, scale, and economics. In particular, enzyme-based workflows are inflexible to different input sample types, have expensive implementation costs, have suboptimal hybrid capture, and have biased fragmentation that can affect downstream results and ultimately increase sequencing costs (Figure 1). Covaris’ cutting-edge AFA technology, the gold-standard in mechanical fragmentation, can help optimize workflows, ensure superior performance, simplify processes, and save significant time and money. With Covaris, researchers can confidently detect and assess genetic risks with precision and accuracy.

Figure 1. The Covaris Germline Solution. Covaris DNA fragmentation produces high quality, reliable sequencing performance and higher throughput, while saving on costs.

Covaris’ DNA and RNA shearing solutions are designed to seamlessly integrate with a variety of library prep kits for whole genome, whole exome, and targeted panel workflows on various sequencing platforms. The established procedures not only cut down time spent on optimization but are also easy to automate. Covaris fragmentation provides higher quality libraries, reliable sequencing performance, and increased throughput–resulting in cost savings.

Figure 2. Library prep and sequencing compatibility.

Covaris Improves Fragmentation Bias, Duplication Rates, and Library Complexity

In a collaborative effort, Covaris and Dana-Farber Cancer Institute compared the results of leading enzymatic fragmentation with library preparations utilizing Covaris’ ultrasonication technology.

As depicted in Figure 3, the graph on the left shows a noticeable increase in base bias at the DNA rupture point when enzymatic fragmentation is used. This bias is also apparent in the range of duplication rate when PCR-free library preps are used, which can significantly affect downstream analysis and sequencing costs. However, libraries created using mechanical fragmentation exhibit no base bias at the fragmentation point and decrease duplication rate by 20 to 80%, reducing flow cell use and sequencing costs.

Figure 3. Addressing fragmentation bias.

Reproducible DNA Fragmentation Across Varied Sample Types

Further research comparing mechanical and enzymatic fragmentation performance across a variety of input sample types demonstrated that Covaris’ process not only enabled a single workflow to accept all input types, but also yielded consistent fragmented DNA insert sizes, both within and across different DNA origin sample types (Figure 4). Covaris’ approach consistently demonstrated higher robustness and tight control over fragment distribution, which has significant implications for the performance and cost-efficiency of sequencing.

Figure 4. Reproducible DNA fragmentation across mixed sample types.

Uniform DNA Shearing Leads to Improved Exome Enrichment

Data produced in collaboration with Quest Diagnostics (Figure 5) revealed a threefold improvement in exome enrichment when mechanical fragmentation was employed in library preparation. The Covaris solution offered superior control over DNA size.

Figure 5. Uniform DNA shearing leads to improved exome enrichment and improved hybrid capture.

Lack of size control can result in DNA straying off target, leading to subpar coverage. The wider the dispersion, the less efficient the process becomes. Lower enrichment values can lead to either a need for more sequencing or less efficient use of the flow cell, both of which can drastically impact overall costs. With Covaris’ mechanical shearing solution, they observed improved hybrid capture and fewer failures in whole exome sequencing. 3x better fold enrichment also leads to more space on the flow cell, ultimately resulting in more efficient use and cost reduction.

In another case study with Radboud University Medical Center in the Netherlands, the overall performance of a fully automated workflow using Covaris fragmentation for whole exome sequencing in germline NGS was evaluated. With a sample population of 339, a pass rate of 98.2% with coverage of the sample exceeding 80x was shown, which is higher than the average coverage achieved with enzymatic fragmentation-based library prep methods (Figure 6).

Figure 6. Automated and streamlined library prep with 98.2% sample pass rate.

Conclusion

The rapid advancements being made in sample prep demand that labs stay ahead with the most advanced technologies. Covaris stands at the forefront with sample prep solutions powered by Adaptive Focused Acoustics (AFA) technology. Covaris offers a number of advantages over traditional enzyme fragmentation library prep, including optimized workflows, superior performance, significant time savings, cost-effectiveness, reproducibility, flexibility, and scalability.

Are you interested in learning more about Covaris’ AFA technology? Click here to reach out to one of our specialists.