In a recent article published in the journal Scientific Reports, researchers proposed an automated microfluidic system for preparing sequencing libraries, a key step in next-generation sequencing (NGS) technology. This new system could revolutionize the accessibility and efficiency of NGS-based diagnostics, especially for point-of-care (PoC) sequencing.
Comparison of sequencing performance of off- and on-chip processed samples. Reference cfDNA with different mutational frequencies were used with 10 ng DNA input for targeted enrichment PCR. The 5%/6.3% sample was also processed with only 1 ng DNA input. Dotted lines divide mutations with different frequencies. Image Credit: https://www.nature.com/articles/s41598-024-67950-6
Background
NGS has emerged as a transformative technology in genomics research and clinical diagnostics. It enables high-throughput, cost-effective genetic analysis, providing insights into disease pathogenesis, drug response, and evolutionary relationships. NGS is essential for cancer diagnostics, allowing the detection of important mutations for therapy decisions. In contrast, regular screenings can quickly identify therapy resistances by monitoring tumor progression.
Traditional biopsies require surgical intervention and are not suitable for frequent sampling. In contrast, liquid biopsies, such as blood samples, are non-invasive. The high sensitivity of NGS is necessary to detect events like mutations in cell-free DNA (cfDNA) from tumors. However, the library preparation process, which involves enzymatic reactions and purification steps, is complex, time-consuming, and prone to contamination. This complexity has hindered the widespread adoption of NGS in clinical settings, especially in resource-limited environments.
About the Research
In this paper, the authors aimed to address the challenges of manual library preparation by developing an automated microfluidic system. They used the commercially available Vivalytic lab-on-a-chip (LoC) platform from Bosch Healthcare Solutions GmbH to integrate the entire library preparation workflow into a compact, user-friendly device.
The Vivalytic system consists of a pneumatic unit to control the microfluidics, a thermal system with 12 heaters, and an optical readout system for quantitative PCR (qPCRs) and microarrays. The cartridge, the heart of the system, has four layers combined by laser welding. The fluidic and pneumatic layers are separated by a thermoplastic polyurethane (TPU) membrane, enabling actuation.
The researchers designed and developed a multiplex PCR targeting various tumor-associated single nucleotide variants (SNVs) of cfDNA material with varying allelic frequencies. This allowed them to compare the performance of the automated (on-chip) library preparation to the manual (off-chip) workflow.
The study implemented a targeted sequencing library preparation workflow, incorporating enzyme reactions for PCR, ligation, end-repair, and enrichment PCR into two cartridges. They then applied a short DNA purification process using carboxylated magnetic beads with solid-phase reversible immobilization. Furthermore, quantification was integrated separately to prepare the sample for the sequencing library.
Research Findings
The outcomes showed that the automated on-chip library preparation performed comparably to the manual off-chip workflow in terms of amplicon distribution and detection of low-frequency mutations. The authors detected mutations with frequencies ranging from 0.1 % to 6.3 % using both approaches. They highlighted the importance of optimizing the PCR cycling parameters for the on-chip system.
The on-chip quantification module using an intercalating dye and an automated serial dilution system accurately determined DNA concentration, even for highly concentrated samples, by identifying the optimal dilution step within the linear range of the assay. When comparing the sequencing performance of the on-chip and off-chip prepared libraries, the researchers found a strong correlation (Pearson correlation coefficient of 0.94) between the two approaches. The mutation frequencies from 5 %/6.3 % down to 0.1 %/0.13 % could be detected with similar accuracy, regardless of the processing method.
The researchers found that shuttling between temperature zones in the microfluidic platform did not affect the target enrichment of a multiplex PCR. They successfully integrated a DNA quantification module into the microfluidic system, with an efficiency of around 50 % compared to the off-chip reference. This module uses an automated serial dilution method to determine the library concentration, which is crucial for optimal sequencing performance. The study optimized the purification using a peristaltic pump mechanism to minimize bead loss.
Applications
The novel library preparation system has the potential to revolutionize NGS-based diagnostics by bringing the technology closer to the point of care. By integrating the entire workflow into a compact, user-friendly device, the system could enable small hospitals, ambulatory healthcare centers, and doctor's offices to utilize NGS for diagnostic and therapy monitoring purposes.
The system's flexibility, which allows for both targeted and universal whole-genome sequencing approaches, further expands its potential applications. The ability to customize the library preparation workflow, such as using different multiplex PCR panels or index PCR beads, makes the system adaptable to various diagnostic needs.
Conclusion
In summary, integrating a customizable library preparation workflow into a microfluidic LoC platform marked a major step toward making point-of-care sequencing a reality. This automated, low-cost, and flexible system enabled broader use of NGS in small healthcare settings, improving access to personalized diagnostics and therapy monitoring.
By addressing the challenges of manual library preparation, this system enhanced the accessibility and efficiency of NGS-based diagnostics, benefiting both patients and healthcare providers. Simplifying the library preparation process transformed this technology for diagnosing and treating diseases, ultimately improving patient outcomes.
Moving forward, the authors suggested several improvements, including reducing the number of cartridges, implementing a variable system for universal and targeted approaches, optimizing magnetic bead purification, and developing a DNA extraction module. These enhancements could further streamline the process and expand the capabilities of the system.
Journal Reference
Hoffmann, A., Timm, A., Johnson, C. et al. Automation of customizable library preparation for next-generation sequencing into an open microfluidic platform. Sci Rep 14, 17150 (2024). DOI: 10.1038/s41598-024-67950-6, https://www.nature.com/articles/s41598-024-67950-6
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