Do we still need Illumina sequencing data?: Evaluating Oxford Nanopore Technologies R10.4.1 flow cells and v14 library prep kits for Gram negative bacteria whole genome assemblies

The best whole genome assemblies are currently built from a combination of highly accurate short-read sequencing data and long-read sequencing data that can bridge repetitive and problematic regions. Oxford Nanopore Technologies (ONT) produce long-read sequencing platforms and they are continually improving their technology to obtain higher-quality read data that is approaching the quality obtained from short-read platforms such as Illumina. As these innovations continue, we were interested in evaluating how much ONT read coverage produced by the Rapid Barcoding Kit v14 (SQK-RBK114) is necessary to generate high-quality hybrid and long-read-only genome assemblies for a panel of carbapenemase-producing Enterobacterales bacterial isolates. We found that 30X long-read coverage is sufficient if Illumina data is available, and that 100X long-read coverage is recommended for long-read-only assemblies. We found that Illumina polishing is still improving SNVs and INDELs in long-read-only assemblies. We also examined if antimicrobial resistance genes could be accurately identified in long-read-only data, and found that Flye assemblies regardless of ONT coverage detected > 94 % of resistance genes at 100% identity and length. Overall, the Rapid Barcoding Kit v14 and long-read-only assemblies can be an optimal sequencing strategy depending on the specific use case and resources available. ### Competing Interest Statement The authors have declared no competing interest.