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Updated: Sep 8, 2025
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Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example
Published on: March 11, 2020
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An open-source nanopore-only sequencing workflow for analysis of clonal outbreaks delivers short-read level accuracy
Nick Vereecke1, Thomas B Yoon1, Ting L Luo2
1Bacterial Pathogenesis and Antimicrobial Resistance Section (BPARS), Laboratory of Clinical Immunology & Microbiology (LCIM), National Institute for Allergy and Infectious Disease (NIAID), National Institutes of Health (NIH), Bethesda, Maryland, USA.
Journal of Clinical Microbiology
|July 18, 2025
Summary
This study optimized a nanopore-only sequencing workflow for bacterial outbreak analysis, achieving results comparable to traditional short-read methods. This open-source approach enhances hospital infection control by enabling rapid and accurate pathogen identification.
Area of Science:
- Genomics
- Infectious Disease Epidemiology
- Bioinformatics
Background:
- Short-read sequencing has been the standard for bacterial whole-genome sequencing and outbreak tracing.
- Long-read sequencing platforms like Oxford Nanopore Technologies (ONT) offer advantages in cost, portability, and speed.
- Historically, ONT's higher basecall error rates limited its clinical microbiology applications, including outbreak investigations.
Purpose of the Study:
- To optimize a streamlined, nanopore-only sequencing workflow for the epidemiologic analysis of bacterial pathogens.
- To validate the performance of this workflow against established short-read sequencing methods for outbreak tracing.
- To enable broader implementation of ONT-only genomes and core genome multilocus sequence typing (cgMLST) for global hospital outbreak investigations.
Main Methods:
- Optimized a modified rapid barcoding library preparation strategy with temperature ramps for high-GC content genomes.
- Benchmarked the performance of the dorado suite (v0.9.1) for basecalling, error correction, and polishing.
- Utilized Flye for long-read assembly and pyMLST for cgMLST analysis.
- Compared nanopore-only results with Illumina short-read sequencing data for accuracy assessment.
Main Results:
- Optimal performance achieved using dorado sup@v5.0.0 basecalling with integrated error correction and bacterial model polishing.
- Nanopore-only assemblies demonstrated fully concordant cgMLST-based minimum spanning trees compared to short-read references.
- Whole-genome analysis showed high concordance, with as few as two discordant positions per genome compared to short-read assemblies.
- The workflow was successfully validated on diverse clinical outbreak isolates including *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, *Enterococcus faecium*, and *Staphylococcus aureus*.
Conclusions:
- The optimized nanopore-only workflow provides accurate and reliable results for bacterial clonality and outbreak analysis.
- This open-source workflow offers performance comparable to Illumina short-read sequencing, facilitating rapid hospital infection control.
- The streamlined approach enhances the feasibility of using ONT-only sequencing for routine clinical microbiology and epidemiological surveillance.
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