A gel-less, bead-less genomic size selection approach on a rapid and inexpensive microfluidic sample preparation platform

Abstract Long-read sequencing is revolutionizing genomics by enabling the analysis of much larger DNA fragments (>10 kb), unlike traditional short-read sequencing that processes smaller fragments. This capability resolves complex genomic regions such as repetitive sequences, structural variations, and long-range haplotypes often inaccessible with short reads. Long-read technologies, such as PacBio and Oxford Nanopore (ONT), excel in capturing complex genomic features but are most impactful when processing larger DNA molecules. As these methods improve in cost and accuracy, their adoption is poised to accelerate, advancing our understanding of genomic architecture and its relevance to health and disease. However, sample prep, particularly size selection, remains a major bottleneck for both sequencing methods. Current techniques, relying on beads and gels, are labor-intensive, error-prone, and costly, heightening risks of sample loss and contamination. Accurate size selection is crucial, especially for PacBio, where fragment sizes under 10 kb compromise performance. To address this, MicroPure Genomics Inc. proposes an automated, bead-less, gel-less microfluidic size selection approach. This innovation simplifies workflows, reduces costs, improves reproducibility, and enhances sequencing performance, paving the way for accurate and comprehensive genomic analyses. Using patented Electro-Hydrodynamic (EH) Trapping technology, the μPrep enables gentle purification and microfluidic handling without beads, minimizing sample degradation. Validated for ONT protocols, μPrep reduces prep time (>50%), costs (>30%), and complexity (>90%), while improving accuracy. These advancements promise to expand long-read sequencing adoption, driving genomic research and clinical applications. This Phase I proposal seeks to extend μPrep's capabilities to support PacBio, whose high-throughput Revio sequencer demands precise sample prep. We will develop and integrate EH-based size selection for PacBio workflows, targeting genomic fragments (18 kb and PacBio-prepared DNA). Strong preliminary results will guide efforts to demonstrate feasibility, streamline workflows, and enhance compatibility with existing platforms. Specific aims include: 1. Development of the EH Trapping for genomic size separation and selection. 2. Size selection for 18 kb DNA. 3. Size selection of PacBio library-prepared DNA using EH Trapping. Phase II will fully integrate the sample prep process into a scalable cartridge, facilitating commercial validation and adoption. Ultimately, this innovation will enhance sequencing workflows and accelerate breakthroughs in genomic science and clinical diagnostics.