Automation of customizable library preparation for next-generation sequencing into an open microfluidic platform

Area of Science:

• Molecular Biology
• Biotechnology
• Medical Diagnostics

Background:

• Next-generation sequencing (NGS) is crucial for cancer management, particularly for monitoring therapy response using cell-free DNA (cfDNA) due to its high sensitivity in detecting rare mutations.
• Traditional NGS library preparation is complex and time-consuming, often requiring expensive, large-scale automation unsuitable for low-to-medium throughput labs.
• Existing automation solutions are costly and require expert handling, limiting accessibility for smaller laboratories.

Purpose of the Study:

• To present a proof-of-concept for an automated, low-to-medium throughput library preparation workflow for NGS.
• To demonstrate an alternative automation solution using a commercially available, open lab-on-a-chip platform.
• To optimize common library preparation steps within a microfluidic environment for cfDNA analysis.

Main Methods:

• Developed a lab-on-a-chip cartridge integrating customizable PCR for target enrichment, end-repair, adapter ligation, magnetic bead-based nucleic acid purification, and quantification.
• Optimized standard library preparation steps for a microfluidic environment.
• Validated the workflow using reference cfDNA with known mutations at varying allelic frequencies.

Main Results:

• The automated lab-on-a-chip workflow successfully performed all key library preparation steps.
• Amplicon sequencing results from the automated system showed high comparability with manual processing (Pearson r = 0.94).
• The system demonstrated functionality with cfDNA containing known mutations at different allelic frequencies.

Conclusions:

• The proposed automated lab-on-a-chip workflow offers a viable and cost-effective alternative for NGS library preparation in low-to-medium throughput settings.
• This microfluidic approach can facilitate the integration of NGS into routine cancer management by simplifying and automating library preparation.
• The technology has the potential to broaden the application of NGS beyond research into clinical diagnostics for cancer therapy monitoring.