Clinical validation of an optimised metagenomic nanopore sequencing method for detecting viral respiratory pathogens
7 affiliations
- 1NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, UK; Modernising Medical Microbiology Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Correspondence to: NDM Experimental Medicine Division, John Radcliffe Hospital, Headley Way, Headington, Oxford OX3 9DU, UK.
- 2NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, UK; Modernising Medical Microbiology Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK
- 3Modernising Medical Microbiology Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK
- 4Oxford University Hospitals NHS Foundation Trust, Oxford, UK
- 5NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, UK; Modernising Medical Microbiology Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK; NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford, Oxford, UK
- 6NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, UK; Modernising Medical Microbiology Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Oxford University Hospitals NHS Foundation Trust, Oxford, UK; NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at University of Oxford, Oxford, UK
- 7NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford, UK; Modernising Medical Microbiology Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK; Oxford University Hospitals NHS Foundation Trust, Oxford, UK
Abstract
Background: Clinical metagenomics (CMg) offers high-throughput respiratory pathogen detection with a wider range than targeted, probe-dependent diagnostics. Sequencing cost and the challenges of high host biomass are barriers to the use of CMg in high-throughput respiratory pathogen detection in non-invasive samples such as nasopharyngeal swabs. Methods: We optimised a nanopore sequencing workflow to detect RNA viruses in nasopharyngeal swabs, employing pathogen enrichment, by background nucleic acid depletion and SISPA amplification, and Oxford Nanopore Technology (ONT) sequencing. As a pre-requisite for agnostic pathogen detection, we first derived quality control (QC) criteria and diagnostic thresholds against a gold-standard comprising 23 pathogen targets detected by routine multiplex PCR. In this diagnostic accuracy study we validated this workflow using 344 prospectively collected upper respiratory tract samples submitted for routine testing. Findings: Using pre-defined QC and positivity criteria, the workflow’s sensitivity versus PCR was 51% (95%CI: 45%–57%) (133/260 positive targets detected) (ranging from 19%–85% across pathogens with >20 gold- standard detections), and specificity 99.8% (95%CI: 99.6%–99.9%) (3836/3845 negative targets not detected). Sensitivity improved to 58% (159/274) using post-hoc optimised thresholds, 61% (159/260) only considering RNA pathogens, 70% (144/207) excluding rhinovirus/enterovirus and 83% (140/169) excluding samples with post workflow qPCR Ct values ≥35. Read crossover from multiplex sequencing contributed most (7/9) false-positives. Only 2 plausible additional pathogens were identified (rhinovirus and coronavirus OC43). 41 respiratory syncytial virus (RSV), 13 influenza A and 10 rhinovirus/enterovirus were successfully sub-typed by sequencing. Multiplexed nanopore sequencing costs were £112/sample. Interpretation: Although CMg has substantial diagnostic potential, this validation study demonstrates the technical limitations of current metagenomic sequencing methods applied to viral detection in upper respiratory tract samples with high host and low pathogen biomass. Its greater sensitivity at higher viral loads demonstrates the importance of identifying the most appropriate use cases to maximise its utility and value.
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BibTeX
@article{sanderson2026clinical,
title = {{Clinical validation of an optimised metagenomic nanopore sequencing method for detecting viral respiratory pathogens}},
author = {Nicholas D. Sanderson and Kate E. Dingle and Katie M.V. Hopkins and Ali Vaughan and Matthew Colpus and Melody Parker and Elisabeth Vilde Dietz and Jessica Gentry and Anita Justice and Sarah Oakley and Lucinda Barrett and T. Phuong Quan and Nicole Stoesser and David W. Eyre and Philip Bejon and Ann Sarah Walker and Bernadette C. Young},
journal = {Journal of Infection},
year = {2026},
month = oct,
doi = {10.1016/j.jinf.2026.106839},
url = {https://doi.org/10.1016/j.jinf.2026.106839},
note = {CC BY},
}
RIS
TY - JOUR
TI - Clinical validation of an optimised metagenomic nanopore sequencing method for detecting viral respiratory pathogens
AU - Nicholas D. Sanderson
AU - Kate E. Dingle
AU - Katie M.V. Hopkins
AU - Ali Vaughan
AU - Matthew Colpus
AU - Melody Parker
AU - Elisabeth Vilde Dietz
AU - Jessica Gentry
AU - Anita Justice
AU - Sarah Oakley
AU - Lucinda Barrett
AU - T. Phuong Quan
AU - Nicole Stoesser
AU - David W. Eyre
AU - Philip Bejon
AU - Ann Sarah Walker
AU - Bernadette C. Young
PY - 2026
DA - 2026/10/23
JO - Journal of Infection
DO - 10.1016/j.jinf.2026.106839
UR - https://doi.org/10.1016/j.jinf.2026.106839
AB - Background: Clinical metagenomics (CMg) offers high-throughput respiratory pathogen detection with a wider range than targeted, probe-dependent diagnostics. Sequencing cost and the challenges of high host biomass are barriers to the use of CMg in high-throughput respiratory pathogen detection in non-invasive samples such as nasopharyngeal swabs. Methods: We optimised a nanopore sequencing workflow to detect RNA viruses in nasopharyngeal swabs, employing pathogen enrichment, by background nucleic acid depletion and SISPA amplification, and Oxford Nanopore Technology (ONT) sequencing. As a pre-requisite for agnostic pathogen detection, we first derived quality control (QC) criteria and diagnostic thresholds against a gold-standard comprising 23 pathogen targets detected by routine multiplex PCR. In this diagnostic accuracy study we validated this workflow using 344 prospectively collected upper respiratory tract samples submitted for routine testing. Findings: Using pre-defined QC and positivity criteria, the workflow’s sensitivity versus PCR was 51% (95%CI: 45%–57%) (133/260 positive targets detected) (ranging from 19%–85% across pathogens with >20 gold- standard detections), and specificity 99.8% (95%CI: 99.6%–99.9%) (3836/3845 negative targets not detected). Sensitivity improved to 58% (159/274) using post-hoc optimised thresholds, 61% (159/260) only considering RNA pathogens, 70% (144/207) excluding rhinovirus/enterovirus and 83% (140/169) excluding samples with post workflow qPCR Ct values ≥35. Read crossover from multiplex sequencing contributed most (7/9) false-positives. Only 2 plausible additional pathogens were identified (rhinovirus and coronavirus OC43). 41 respiratory syncytial virus (RSV), 13 influenza A and 10 rhinovirus/enterovirus were successfully sub-typed by sequencing. Multiplexed nanopore sequencing costs were £112/sample. Interpretation: Although CMg has substantial diagnostic potential, this validation study demonstrates the technical limitations of current metagenomic sequencing methods applied to viral detection in upper respiratory tract samples with high host and low pathogen biomass. Its greater sensitivity at higher viral loads demonstrates the importance of identifying the most appropriate use cases to maximise its utility and value.
SN - 1532-2742
ER -