RT Journal
A1 Cahyani, Inswasti
A1 Tyson, John
A1 Holmes, Nadine
A1 Quick, Josh
A1 Moore, Chris
A1 Loman, Nick
A1 Loose, Matthew
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 May 01 
VO 35 
IS 5 
SP 1154 
OP 1166 
DO 10.1101/gr.279943.124 
UL http://genome.cshlp.org/content/35/5/1154.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 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.