RT Journal
A1 Cahyani, Inswasti
A1 Tyson, John
A1 Holmes, Nadine
A1 Quick, Joshua
A1 Moore, Chris
A1 Loman, Nicholas James
A1 Loose, Matt
T1 An optimized toolkit for high molecular weight DNA extraction and ultra-long-read nanopore sequencing using glass beads and hexamminecobalt(III) chloride
JF Genome Research 
JO Genome Research 
YR 2025 
FD March 24 
DO 10.1101/gr.279943.124 
SP gr.279943.124 
UL http://genome.cshlp.org/content/early/2025/03/21/gr.279943.124.abstract 
AB 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 clean-up 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 to 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.