A proficiency testing of metagenomics as a diagnostic tool for the detection of RNA viruses in swine fecal material

Metagenomics next-generation sequencing (mNGS) is widely used as a generic molecular diagnostic tool for detection of the unknown, emerging, or re-emerging pathogens. A typical methodological workflow includes sample disintegration, nucleic acid extraction, double strand cDNA synthesis in case of RNA, library preparation, and data analysis, all of which affect the performance of mNGS diagnostics. This study was undertaken among six European laboratories within METASTAVA One Health European Joint Programme. Modules suitable to build standardized mNGS workflows were selected and tested. For each of the modules, two methods and common procedures for analysis of sequencing data were chosen. To finally evaluate the selected modules in terms of (i) suitability to detect viruses in native samples and (ii) simultaneously assess their reproducibility, a proficiency test (PT) was carried out as part of METASTAVA. For this PT, a pre-analysed swine faecal sample containing a porcine astrovirus and RNA extracted from that sample were distributed among the participants. Most importantly, using the abovementioned methods comprised in the METASTAVA modular mNGS workflow, all participants identified the same viral species belonging to the family Astroviridae in both, the clinical sample and in the distributed RNA. In detail, all participants classified the majority of viral reads to two species of porcine astroviruses (PAstV2 and PAstV4), regardless of the used analysis method (Kraken and Bowtie2). Moreover, participants performed well in terms of repeatability when the faecal sample was tested in duplicate, resulting in a low coefficient of variation. Therefore, this PT showed that the methods selected as METASTAVA mNGS modules are (i) well suited for virus identification even from challenging matrices like faeces and (ii) the selected methods can reproducibly be applied.

宏基因组下一代测序（Metagenomics next-generation sequencing，简称mNGS）被广泛用作通用分子诊断工具，用于检测未知、新发或再现性病原体。典型的方法学流程涵盖样本裂解、核酸提取、针对RNA样本的双链互补DNA（cDNA）合成、文库制备以及数据分析，所有环节均会影响mNGS诊断的性能。本研究依托METASTAVA One Health欧洲联合计划，在6家欧洲实验室中开展。研究筛选并验证了可用于构建标准化mNGS流程的模块化组件，针对每个组件选取了两种测序数据分析方法及通用分析流程。为最终评估所选模块化组件的两项性能：（i）对天然样本中病毒的检测适配性，（ii）同时评估其重现性，研究作为METASTAVA项目的一部分开展了能力验证（Proficiency Test，简称PT）。本次能力验证所用的分发样本包括：一份预先处理的猪粪便样本（含猪星状病毒）以及从该样本中提取的RNA。尤为重要的是，所有参与实验室均采用METASTAVA模块化mNGS流程中包含的上述分析方法，在临床样本与分发的RNA样本中均鉴定出了星状病毒科（Astroviridae）的同一病毒物种。具体而言，无论采用何种分析方法（Kraken与Bowtie2），所有参与实验室均将大部分病毒测序读段（reads）归类为两种猪星状病毒：猪星状病毒2型（PAstV2）与猪星状病毒4型（PAstV4）。此外，当对粪便样本进行重复检测时，所有参与实验室的重复性表现优异，变异系数较低。综上，本次能力验证结果表明：（i）所选METASTAVA mNGS模块化组件即便针对粪便这类复杂基质样本，也可良好适配病毒鉴定工作；（ii）所选方法可实现稳定重现的应用效果。