How good is good enough?

Abstract With the release of the R10 Flow Cell in 2019 and the Dorado basecaller in 2023, long-read nanopore sequencing has displayed a significant improvement in accuracy. Since then, there have been studies investigating the basecalling runtime, output read quality, methylation detection accuracy, and nanoporeonly assembly quality. However, these studies have been limited to small quantities of organisms and have not explored the general-use cases that may apply to many laboratories. How much better performance does Dorado offer over Guppy at the various accuracy modes in relation to the time spent basecalling for as many diverse organism types as possible? While at the time of writing this abstract, the analysis is ongoing, the study described here will evaluate 283 different GridION generated sequencing samples across bacterial, fungal, and viral species in terms of basecalling speed and average read quality for both Guppy and Dorado with each using the fast, HAC, and SUP accuracy modes. Further, both nanopore-only and Illumina/Oxford Nanopore hybrid assemblies from the resultant six collections of data will be evaluated for completion with respect to taxonomic domain, GC content, assembly N50, genome size, replicon count, small variants, and gene content. To date, this represents the first time such a broad and multi-method study has been performed — further underscored by performing all sequencing by the same user, instrument, chemistry, flow cell, laboratory, and physical basecalling hardware. This study aims to guide sequencing laboratories on the effects of the options available. Biography David Yarmosh is a lead bioinformatician in the ATCC Sequencing and Bioinformatics Center. His work with Oxford Nanopore Technologies data began with real-time metagenomic sequencing analysis, researching a method to identify organisms within 24 hours of sampling in a field-forward setting. He built a small toolkit that took advantage of the MinION streaming output to produce taxonomic identification as reads were generated. He has since published broadscale interplatform SARS-COV-2 variant determination and reference-grade hybrid microbial assembly. His interests lie in identifying limitations within data, hardware, and software and finding optimizations that suit the constraints of projects, budgets, and time.