Accurate and reproducible whole-genome genotyping for bacterial genomic surveillance with Nanopore sequencing data

Area of Science:

• Genomics
• Bioinformatics
• Microbiology

Background:

• Oxford Nanopore Technologies (ONT) sequencing offers cost-effective, real-time genomic data but historically lacked the accuracy for fine-scale bacterial analysis.
• Multidrug-resistant organisms (MDROs) pose a significant public health threat, necessitating accurate and reproducible genomic surveillance methods.

Purpose of the Study:

• To evaluate recent advancements in ONT basecalling and polishing software, alongside the ONT-cgMLST-Polisher, for improving bacterial genomic analysis.
• To assess the accuracy and reproducibility of ONT sequencing for bacterial genomic surveillance, particularly for MDROs.

Main Methods:

• Benchmarking ONT long-read sequences against Illumina short-read and hybrid "ground truth" assemblies for 80 MDROs.
• Utilizing Dorado Super Accurate (SUP) models (v.4.3, v.5.0) for basecalling, Flye for assembly, and Medaka (v.1.12, v.2.0) for polishing.
• Applying the ONT-cgMLST-Polisher tool to further refine consensus sequences and evaluating results through a multi-laboratory ring trial.

Main Results:

• ONT sequencing accuracy, measured by cgMLST allele distance (AD) to ground truth, significantly improved with newer Dorado SUP models and Medaka v.2.0.
• The ONT-cgMLST-Polisher further reduced AD to an average of 0.09 with SUP m4.3 and 0.04 with SUP m5.0.
• Ring trial data confirmed improved reproducibility with SUP m5.0, achieving average ADs of 0.36 and 0.17 (with polisher).

Conclusions:

• Recent ONT software improvements (Dorado, Medaka) and the ONT-cgMLST-Polisher enhance sequencing accuracy and reproducibility for bacterial genomics.
• These advancements make ONT whole-genome sequencing suitable for routine bacterial genomic surveillance, even in resource-limited laboratories.
• Oxford Nanopore Technologies sequencing is now a viable and accurate tool for bacterial genotyping and epidemiological studies.