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

IF 4 3区医学 Q2 VIROLOGY

Journal of Clinical Virology Pub Date : 2024-05-25 DOI:10.1016/j.jcv.2024.105695

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 , Jutte J.C. de Vries

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引用次数: 0

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.

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对临床病毒元基因组学的短读和长读湿实验室方案进行多中心基准测试。

元基因组学正逐渐用于诊断传染病。然而，对病毒检测方案的深入比较仅限于个别实验工作流程和实验室。在本研究中，我们介绍了由欧洲临床病毒学学会（ESCV）NGS 网络（ENNGS）发起的临床诊断实验室使用的元基因组学方案基准。我们设计了一个模拟病毒参考面板，以模拟低生物量临床标本。该面板用于评估 ENNGS 成员机构诊断实验室目前使用的十二种元基因组湿实验室方案的性能。其中包括 Illumina 和 Nanopore、霰弹枪和靶向捕获探针方案。使用中央生物信息学管道对灵敏度、特异性和定量潜力等性能指标进行了评估。总体而言，所有经过评估的元基因组湿实验室方案都能检测到载量低至 104 copies/ml 的病毒病原体（在我们的 PCR 检测中，CT 值为 31）。相比之下，只有少数方案能检测到 CT 值为 35 或更高的低含量混合病毒。将参考组作为金标准，根据水平基因组覆盖率确定了每个方案的最佳阈值，以确定阳性结果。采用这些阈值后，方案的灵敏度和特异性分别为 67% 至 100% 和 87% 至 100%。目前，临床诊断实验室正在使用多种元基因组方案。低含量病毒病原体和混合感染的检测仍然是一项挑战，这意味着临床环境中使用的元基因组分析需要标准化。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Clinical Virology 医学-病毒学

CiteScore

22.70

自引率

1.10%

发文量

149

审稿时长

24 days

期刊介绍： The Journal of Clinical Virology, an esteemed international publication, serves as the official journal for both the Pan American Society for Clinical Virology and The European Society for Clinical Virology. Dedicated to advancing the understanding of human virology in clinical settings, the Journal of Clinical Virology focuses on disseminating research papers and reviews pertaining to the clinical aspects of virology. Its scope encompasses articles discussing diagnostic methodologies and virus-induced clinical conditions, with an emphasis on practicality and relevance to clinical practice. The journal publishes on topics that include: • new diagnostic technologies • nucleic acid amplification and serologic testing • targeted and metagenomic next-generation sequencing • emerging pandemic viral threats • respiratory viruses • transplant viruses • chronic viral infections • cancer-associated viruses • gastrointestinal viruses • central nervous system viruses • one health (excludes animal health)