Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics

F Xavier Lopez-Labrador; Michael Huber; Igor A Sidorov; Julianne R Brown; Lize Cuypers; Lies Laenen; Bert Vanmechelen; Piet Maes; Nicole Fischer; Ian Pichler; Nathaniel Storey; Laura Atkinson; Stefan Schmutz; Verena Kufner; Sander van Boheemen; Claudia E Mulders; Adam Grundhoff; Patrick Blümke; Alexis Robitaille; Ondrej Cinek; Klára Hubáčková; Kees Mourik; Stefan A Boers; Lea Stauber; Maud Salmona; Pierre Cappy; Alban Ramette; Alessandra Franze'; Jerome LeGoff; Eric C J Claas; Christophe Rodriguez; Jutte J C de Vries; European Society of Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS)

doi:10.1016/j.jcv.2024.105695

Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics

J Clin Virol. 2024 Aug:173:105695. doi: 10.1016/j.jcv.2024.105695. Epub 2024 May 25.

Authors

F Xavier Lopez-Labrador¹, Michael Huber², Igor A Sidorov³, Julianne R Brown⁴, Lize Cuypers⁵, Lies Laenen⁵, Bert Vanmechelen⁶, Piet Maes⁶, Nicole Fischer⁷, Ian Pichler², Nathaniel Storey⁴, Laura Atkinson⁴, Stefan Schmutz², Verena Kufner², Sander van Boheemen⁸, Claudia E Mulders⁸, Adam Grundhoff⁹, Patrick Blümke⁹, Alexis Robitaille⁹, Ondrej Cinek¹⁰, Klára Hubáčková¹⁰, Kees Mourik³, Stefan A Boers³, Lea Stauber¹¹, Maud Salmona¹², Pierre Cappy¹³, Alban Ramette¹¹, Alessandra Franze'¹⁴, Jerome LeGoff¹², Eric C J Claas³, Christophe Rodriguez¹³, Jutte J C de Vries¹⁵; European Society of Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS)

Affiliations

¹ Virology Laboratory, Genomics and Health Area, Center for Public Health Research (FISABIO-Public Health), Generalitat Valenciana, Valencia, Spain; Microbiology & Ecology Department, Medical School, University of Valencia, Spain; and CIBERESP, Instituto de Salud Carlos III, Spain.
² Institute of Medical Virology, University of Zurich, Switzerland.
³ Clinical Microbiological Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands.
⁴ Microbiology, Virology and Infection Prevention & Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, United Kingdom.
⁵ Department of Laboratory Medicine, University Hospitals Leuven, and Laboratory of Clinical Microbiology, KU, Leuven, Belgium.
⁶ Laboratory of Clinical and Epidemiological Virology, Department of Microbiology, Immunology and Transplantation, Rega Institute for Medical Research, KU Leuven, Belgium.
⁷ University Medical Center Hamburg-Eppendorf, UKE Institute for Medical Microbiology, Virology and Hygiene, Germany.
⁸ Viroscience, Erasmus Medical Center, Rotterdam, the Netherlands.
⁹ Leibniz Institute for Virology, Hamburg, Germany.
¹⁰ Department of Medical Microbiology, 2nd Faculty of Medicine, Charles University, and University Hospital Motol, Prague, Czech Republic.
¹¹ Institute for Infectious Diseases, University of Bern, Switzerland.
¹² Virology Department, AP-HP, Hôpital Saint Louis, F-75010 Paris, France.
¹³ Hospital Henri Mondor, Paris, France.
¹⁴ Virology Laboratory, Genomics and Health Area, Center for Public Health Research (FISABIO-Public Health), Generalitat Valenciana, Valencia, Spain.
¹⁵ Clinical Microbiological Laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, the Netherlands. Electronic address: [email protected].

PMID: 38823290
DOI: 10.1016/j.jcv.2024.105695

Abstract

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 10⁴ copies/ml (corresponding to C_T values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of C_T values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

Keywords: Benchmark; Clinical viral metagenomics; Wet lab protocols.

Publication types

Multicenter Study
Research Support, Non-U.S. Gov't

MeSH terms

Benchmarking*
Computational Biology / methods
High-Throughput Nucleotide Sequencing / methods
High-Throughput Nucleotide Sequencing / standards
Humans
Metagenomics* / methods
Metagenomics* / standards
Sensitivity and Specificity*
Virus Diseases / diagnosis
Virus Diseases / virology
Viruses* / classification
Viruses* / genetics
Viruses* / isolation & purification