High intra-laboratory reproducibility of nanopore sequencing in bacterial species underscores advances in its accuracy.

Nanopore sequencing is a third-generation technology known for its portability, real-time analysis and ability to generate long reads. It has great potential for use in clinical diagnostics, but thorough validation is required to address accuracy concerns and ensure reliable and reproducible results. In this study, we automated an open-source workflow (freely available at https://gitlab.com/FLI_Bioinfo/nanobacta) for the assembly of Oxford Nanopore sequencing data and used it to investigate the reproducibility of assembly results under consistent conditions. We used a benchmark dataset of five bacterial reference strains and generated eight technical sequencing replicates of the same DNA using the Ligation and Rapid Barcoding kits together with the Flongle and MinION flow cells. We assessed reproducibility by measuring discrepancies such as substitution and insertion/deletion errors, analysing plasmid recovery results and examining genetic markers and clustering information. We compared the results of genome assemblies with and without short-read polishing. Our results show an average reproducibility accuracy of 99.999955% for nanopore-only assemblies and 99.999996% when the short reads were used for polishing. The genomic analysis results were highly reproducible for the nanopore-only assemblies without short read in the following areas: identification of genetic markers for antimicrobial resistance and virulence, classical MLST, taxonomic classification, genome completeness and contamination analysis. Interestingly, the clustering information results from the core genome SNP and core genome MLST analyses were also highly reproducible for the nanopore-only assemblies, with pairwise differences of up to two allele differences in core genome MLST and two SNPs in core genome SNP across replicates. After polishing the assemblies with short reads, the pairwise differences for cgMLST were 0 and for cgSNP were 0-1 SNP across replicates. These results highlight the advances in sequencing accuracy of nanopore data without the use of short reads.