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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 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 values of 31 in our 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.
Competing Interest Statement
The authors have declared no competing interest.
Funding Statement
This work was partially funded by the European Society of Clinical Virology (ESCV). The work was locally funded at the Leibniz Institute for Virology. This work was locally funded at the University of Zurich by the Clinical Research Priority Program Comprehensive Genomic Pathogen Detection. O.C. and K.H. were financed by National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103 funded by the European Union, Next Generation EU). F.X.L.L is supported by the CIBERESP Network of Excellence, Instituto de Salud Carlos III, Spain.
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I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).
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Footnotes
↵* Shared first authors
#f.xavier.lopez{at}uv.es, alessandra.franze{at}fisabio.es, f.xavier.lopez{at}uv.es, huber.michael{at}virology.uzh.ch, pichler.ian{at}virology.uzh.ch, schmutz.stefan{at}virology.uzh.ch, kufner.verena{at}virology.uzh.ch, I.A.Sidorov{at}lumc.nl, k.mourik{at}lumc.nl, s.a.boers{at}lumc.nl, E.C.J.Claas{at}lumc.nl, julianne.brown{at}gosh.nhs.uk, Nathaniel.Storey{at}gosh.nhs.uk, lize.cuypers{at}uzleuven.be, lies.laenen{at}uzleuven.be, bert.vanmechelen{at}kuleuven.be, piet.maes{at}kuleuven.be, Electronic address: nfischer{at}uke.de, s.vanboheemen{at}erasmusmc.nl, c.mulders{at}erasmusmc.nl, adam.grundhoff{at}leibniz-liv.de, patrick.bluemke{at}leibniz-liv.de, alexis.robitaille{at}leibniz-liv.de, ondrej.cinek{at}lfmotol.cuni.cz, klara.hubackovalf{at}motol.cuni.cz, lea.stauber{at}unibe.ch, alban.ramette{at}unibe.ch, maud.salmona{at}aphp.fr, jerome.le-goff{at}aphp.fr, christophe.rodriguez{at}aphp.fr, pierre.cappy{at}aphp.fr
Data Availability
All data produced in the present study are available upon reasonable request to the authors
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