Over the last 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 is still challenging dependent on sample and library preparation methods. We developed FindingNemo, a toolkit to optimize extractions and ultra-long library clean-up. The protocol can deliver >20 Gb of reads with an N50 > 100kb on a single MinION flow cell or >100Gb of reads on PromethION devices. The method works with multiple sequencing chemistries from R9.4 to R10.4 and, likely, beyond. Our comparisons show there are multiple routes to achieve ultra long library preparations and different approaches may help with challenging and complex samples. We also emphasize the importance of precise determination of input DNA amount through cell counting and quantification.
Nivesh is a molecular biologist leading ONT sequencing efforts at the Vertebrate Genome Lab and is an expert in U/HMW DNA extractions, Bionano optical mapping, and HiFi PacBio sequencing.
Patrick Grady obtained his PhD in Molecular & Cellular Biology under Rachel O’Neill's supervision at the University of Connecticut. His doctoral research centered on the genomic evolution of marsupials and developing bioinformatic techniques to improve telomere-to-telomere whole-genome comparisons.
Alongside his dissertation, Patrick collaborated with the CHM13-T2T Consortium to annotate repeats in the new human genome, developed algorithms to enhance nanopore sequencing analysis, contributed to the Ruminant T2T Consortium, and engaged in single-cell developmental biology research. His long-term goal is to leverage the power of complete T2T genomes alongside functional, epigenetic, and topological genomic data to answer complex evolutionary questions. At Colossal Biosciences, Patrick leads genome assembly projects involving species associated with extinct animals and contributes to functional genomics research with the Thylacine team.
