Background Current molecular diagnostics are limited in the number and type of detectable pathogens. Metagenomic next generation sequencing (mNGS) is an emerging, and increasingly feasible, pathogen-agnostic diagnostic approach. Translational barriers prohibit the widespread adoption of this technology in clinical laboratories. We validate an end-to-end mNGS assay for detection of respiratory viruses. Our assay is optimized to reduce turnaround time, lower cost-per-sample, increase throughput, and deploy secure and actionable bioinformatic results. Methods We validated our assay using residual nasopharyngeal swab specimens from Vancouver General Hospital (n = 359), RT-PCR-positive, or negative for Influenza, SARS-CoV-2, and RSV. We quantified sample stability, assay precision, the effect of background nucleic acid levels, and analytical limits of detection. Diagnostic performance metrics were estimated. Results We report that our mNGS assay is highly precise, semi-quantitative, with analytical limits of detection ranging from 103-104 copies/mL. Our assay is highly specific (100%) and sensitive (61.9% Overall: 86.8%; RT-PCR Ct < 30). Multiplexing capabilities enable processing of up to 55-specimens simultaneously on an Oxford Nanopore GridION device, with results reported within 12-hours. Conclusions This study outlines the diagnostic performance and feasibility of mNGS for respiratory viral diagnostics, infection control, and public health surveillance. We addressed translational barriers to widespread mNGS adoption.

中文翻译：

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验证用于呼吸道病毒不可知检测的自动化端到端宏基因组测序测定

背景 当前的分子诊断在可检测病原体的数量和类型方面受到限制。宏基因组下一代测序 (mNGS) 是一种新兴且日益可行的病原体不可知诊断方法。转化障碍阻碍了该技术在临床实验室的广泛采用。我们验证了用于检测呼吸道病毒的端到端 mNGS 检测。我们的检测经过优化，可缩短周转时间、降低每个样本的成本、提高通量并部署安全且可操作的生物信息结果。方法 我们使用来自温哥华综合医院 (n = 359) 的残留鼻咽拭子样本验证了我们的检测方法，这些样本 RT-PCR 呈阳性，或流感、SARS-CoV-2 和 RSV 呈阴性。我们量化了样品稳定性、测定精度、背景核酸水平的影响以及检测的分析限。估计了诊断性能指标。结果 我们报告说，我们的 mNGS 检测是高度精确的半定量检测，分析检测限范围为 103-104 拷贝/mL。我们的测定具有高度特异性 (100%) 和敏感性 (61.9% 总体：86.8%；RT-PCR Ct < 30)。多重功能可在 Oxford Nanopore GridION 设备上同时处理多达 55 个样本，并在 12 小时内报告结果。结论 本研究概述了 mNGS 在呼吸道病毒诊断、感染控制和公共卫生监测方面的诊断性能和可行性。我们解决了广泛采用 mNGS 的转化障碍。