An optimised toolkit for high molecular weight DNA extraction and ultra-long-read nanopore sequencing using glass beads and hexamminecobalt(III) chloride

Cahyani, Inswasti; Tyson, John; Holmes, Nadine; Quick, Josh; Moore, Chris; Loman, Nick; Loose, Matthew

Abstract

Since the advent of long-read sequencing, achieving longer read lengths has been a key goal for many users. Ultra-long-read sets (N50 ≥ 100 kb) produced from Oxford Nanopore sequencers have improved genome assemblies in recent years. However, despite progress in extraction protocols and library preparation methods, ultra-long sequencing remains challenging for many sample types. Here, we compare various methods and introduce the FindingNemo protocol that: (1) optimizes ultra-high-molecular-weight (UHMW) DNA extraction and library cleanup by using glass beads and hexamminecobalt(III) chloride (CoHex), (2) can deliver high ultra-long sequencing yield of >20 Gb of reads from a single MinION flow cell or >100 Gb from PromethION devices (R9.4–R10.4 pore variants), and (3) is scalable to using fewer input cells or lower DNA amounts, with extraction to sequencing possible in a single working day. By comparison, we demonstrate that this protocol surpasses previous methods by enabling precise determination of input DNA quantity and quality through cell counting, sample dilution, and homogenization techniques.

Citation

Cahyani, I., Tyson, J., Holmes, N., Quick, J., Moore, C., Loman, N., & Loose, M. (2025). An optimised toolkit for high molecular weight DNA extraction and ultra-long-read nanopore sequencing using glass beads and hexamminecobalt(III) chloride. Genome Research, 35(5), 1154-1166. https://doi.org/10.1101/gr.279943.124

• Journal Article Type: Article
• Acceptance Date: Mar 6, 2025
• Online Publication Date: Mar 24, 2025
• Publication Date: 2025-05
• Deposit Date: Mar 14, 2025
• Publicly Available Date: Mar 14, 2025
• Journal: Genome Research
• Print ISSN: 1088-9051
• Electronic ISSN: 1549-5469
• Publisher: Cold Spring Harbor Laboratory Press
• Peer Reviewed: Peer Reviewed
• Volume: 35
• Issue: 5
• Pages: 1154-1166
• DOI: https://doi.org/10.1101/gr.279943.124
• Public URL: https://nottingham-repository.worktribe.com/output/46469149
• Publisher URL: https://genome.cshlp.org/content/35/5/1154