A toolkit for ultra-long sequencing on ONT platforms. FindingNemo Toolkit

Since the advent of long read sequencing, users have sought to achieve ever longer read lengths. Ultra-long reads (defined as a read N50 > 100 kb) have enabled highly contiguous genome assemblies across various sample types. Over the past decade developments in extraction protocols and library preparation have significantly improved sequencing yields for ultra-long protocols on Oxford Nanopore Sequencers. However, ultra-long sequencing remains challenging for many sample types and library preparation methods. Here we evaluate and compare a wide range of ultra-long sequencing methods and introduce a glass bead-based protocol (the FindingNemo protocol) to optimize extractions and library clean-up. We demonstrate that Hexamminecobalt(III) chloride (CoHex) can precipitate ultra-long sequencing libraries, resulting in high-throughput and high-occupancy nanopore sequencing runs. This approach can deliver >20 Gb of reads with an N50 > 100kb on a single MinION flow cell or >100 Gb of reads on PromethION devices. It can be tailored to use lower input amounts, with extraction to sequencing possible in a single working day. We have tested it with multiple sequencing kits and both R9.4 to R10.4 pores. Our comparisons show multiple routes to achieve ultra-long library preparations, and that different approaches may aid in sequencing challenging and complex samples. We also emphasize the importance of precise determination and homogenization of input DNA amount by cell counting, quantification and dilution.