Accurately assembling nanopore sequencing data of highly pathogenic bacteria.

Area of Science:

• Genomics
• Bioinformatics
• Microbial Pathogenesis

Background:

• Whole-genome sequencing and bioinformatics are crucial for bacterial genome exploration and outbreak analysis.
• High sequencing accuracy and precise genome assembly are essential for reliable genotyping and genetic marker detection.

Purpose of the Study:

• To assess the utility of Oxford Nanopore Technologies (ONT) R10.4.1 sequencing for genotyping highly pathogenic bacteria with low mutation rates.
• To evaluate different assembly strategies and their impact on genome accuracy.

Main Methods:

• Sequencing of six reference bacterial strains using ONT R10.4.1 chemistry and Illumina.
• Evaluation of various assembly strategies against RefSeq assemblies as ground truth.
• Analysis of publicly available sequencing data from key bacterial pathogens.

Main Results:

• Assembly quality varied by species; Bacillus anthracis achieved near-perfect assembly, while Brucella spp. showed nucleotide differences.
• Perfect genomes were obtained for Klebsiella variicola, Listeria spp., Mycobacterium tuberculosis, Staphylococcus aureus, and Streptococcus pyogenes.
• Errors, particularly within coding sequences and methylation-related, were observed, though methylation-aware models showed improvement. Core-genome Multilocus Sequence Typing (cgMLST) revealed minor allele differences in some species.

Conclusions:

• Nanopore sequencing data assembly quality for pathogenic bacteria is species and method-dependent.
• Despite persistent errors in assemblies, including cgMLST loci, specific tool combinations can yield perfect genomes for outbreak analysis without short-read polishing.