Comparison of Illumina and Oxford Nanopore Technology for genome analysis of Francisella tularensis, Bacillus anthracis, and Brucella suis

Abstract

Background Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. Results As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. Conclusions In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.

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

“…Long-read-only LSKv14 assemblies have been shown to be accurate for detecting acquired antimicrobial resistance genes, virulence factors, and performing cgMLST analysis 17,19 . Equivalent data for RBKv14 has not yet been published, but 21 tested various genera with RBKv10 on R10.3 but found Illumina data resulted in better AMR gene detection and cgMLST, and both the library preparation kit and flow cells have newer versions.…”

Accurate bacterial outbreak tracing with Oxford Nanopore sequencing and reduction of methylation-induced errors

“…Several studies have shared their findings and reported accuracy levels similar to those from short-read data [17], [18]. However, significant discrepancies between Illumina and Nanopore genomes were also observed for some organisms [19].…”

“…Erroneous basecalls occur in some strains but not others and vary by basecaller and sequencing kits We resequenced the genomes of 33 randomly selected K. pneumonia samples (from a total of 114 outbreak-related isolates) using R10.4 and R10.4.1 flowcells, along with the corresponding library preparation kits (henceforth "Kit 12": SQK-NBD112.24 (early access) and "Kit 14": SQK-NBD114.24 (successor)), to examine if the previously documented conflicting statements could be replicated [17]- [19]. We used cgMLST analysis to compare the sequenced genomes against Illumina data.…”

Genomic and morphological characterization of Knufia obscura isolated from the Mars 2020 spacecraft assembly facility

“…Through rigorous phylogenetic analysis, employing MLSA and WGS methodologies, we have verified the phylogenetic affiliation of FJI-L2-BK-P2. The use of both Illumina and Oxford Nanopore Technology (ONT) sequencing methods was not to compare the DNA sequence composition accuracy between these methods, as their consistency is well-established 37 . The short-read sequencing (Illumina) of the ITS region of FJI-L2-BK-P2 showed a 99.46% identity to the corresponding region in the type strain CBS 148926 T that was sequenced using long read sequencing by the ONT.…”