Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics
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 CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT 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.
Related
- Benchmarking and optimisation of bait-capture metagenomics for sequencing of respiratory viruses at scale
- Accurate MAG reconstruction from a complex soil microbiome through combined short- and HiFi long-reads metagenomics
- Hunting for Helminths: short- and long-read shotgun metagenomics for helminth detection in faecal samples
- Benchmarking batch-effect correction in clinical bead-based multiplex immunoassays: a framework and web tool for cytokine and soluble immune checkpoint data
- Dynamic assessment of renal resistive index using point-of-care ultrasonography for early detection of sepsis-induced acute kidney injury: A multicenter clinical trial protocol
BibTeX
@article{lopezlabrador2026multicenter,
title = {{Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics}},
author = {F. Xavier Lopez-Labrador and Michael Huber and Igor Sidorov and Julianne Brown and Lize Cuypers and Lies Laenen and Bert Vanmechelen and Piet Maes and Nicole Fischer and Ian Pichler and Nathaniel Storey and Laura Atkinson and Stefan Schmutz and Verena Kufner and Sander van Boheemen and Claudia Mulders and Adam Grundhoff and Patrick Blümke and Alexis Robitaille and Ondrej Cinek and Klára Hubáčková and Kees Mourik and Stefan Boers and Lea Stauber and Maud Salmona and Pierre Cappy and Alban Ramette and Alessandra Franze’ and Jérôme Legoff and Eric C.J. Claas and Christophe Rodriguez and Jutte J.C. de Vries},
journal = {Journal of Clinical Virology},
year = {2026},
month = oct,
doi = {10.1016/j.jcv.2024.105695},
url = {https://hal.science/hal-05783115v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics
AU - F. Xavier Lopez-Labrador
AU - Michael Huber
AU - Igor Sidorov
AU - Julianne Brown
AU - Lize Cuypers
AU - Lies Laenen
AU - Bert Vanmechelen
AU - Piet Maes
AU - Nicole Fischer
AU - Ian Pichler
AU - Nathaniel Storey
AU - Laura Atkinson
AU - Stefan Schmutz
AU - Verena Kufner
AU - Sander van Boheemen
AU - Claudia Mulders
AU - Adam Grundhoff
AU - Patrick Blümke
AU - Alexis Robitaille
AU - Ondrej Cinek
AU - Klára Hubáčková
AU - Kees Mourik
AU - Stefan Boers
AU - Lea Stauber
AU - Maud Salmona
AU - Pierre Cappy
AU - Alban Ramette
AU - Alessandra Franze’
AU - Jérôme Legoff
AU - Eric C.J. Claas
AU - Christophe Rodriguez
AU - Jutte J.C. de Vries
PY - 2026
DA - 2026/10/07
JO - Journal of Clinical Virology
DO - 10.1016/j.jcv.2024.105695
UR - https://hal.science/hal-05783115v1
AB - 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 CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT 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.
SN - 1386-6532
SN - 1873-5967
ER -