SARSeq, a robust and highly multiplexed NGS assay for parallel detection of SRAS-CoV2 and other respiratory infections

During a pandemic, mitigation as well as protection of system-critical or vulnerable institutions requires massively parallel, yet cost-effective testing to monitor the spread of agents such as the current SARS-CoV2 virus. Here we present SARSeq, saliva analysis by RNA sequencing, as an approach to monitor presence of SARS-CoV2 and other respiratory viruses performed on tens of thousands of samples in parallel. SARSeq is based on next generation sequencing of multiple amplicons generated in parallel in a multiplexed RT-PCR reaction. It relies on a two-dimensional unique dual indexing strategy using four indices in total, for unambiguous and scalable assignment of reads to individual samples. We calibrated this method using dilutions of synthetic RNA and virions to show sensitivity down to a few molecules, and applied it to hundreds of patient samples validating robust performance across various sample types. Double blinded benchmarking to gold-standard quantitative RT-PCR performed in a clinical setting and a human diagnostics laboratory showed robust performance up to a Ct of 36. The false positive rate, likely due to cross contamination during sample pipetting, was estimated at 0.04-0.1%. In addition to SARS-CoV2, SARSeq detects Influenza A and B viruses as well as human rhinovirus and can be easily expanded to include detection of other pathogens. In sum, SARSeq is an ideal platform for differential diagnostic of respiratory diseases at a scale, as is required during a pandemic. Massively parallel, cost-effective detection of SARS-CoV2 using saliva analysis by RNA sequencing (SARSeq). Study includes left-over patient samples. [contributor] Vienna Covid-19 Detection Initiative Members

在大流行期间，为落实疫情防控举措并保护系统关键或脆弱的公共机构，需要开展大规模并行且兼具成本效益的检测工作，以监测如当前新型冠状病毒（SARS-CoV2）这类病原体的传播态势。本研究介绍了一种名为SARSeq的检测方法：通过RNA测序对唾液样本进行分析，可并行对数以万计的样本开展检测，用于监测SARS-CoV2与其他呼吸道病毒的感染情况。该方法依托多重逆转录聚合酶链式反应（reverse transcription polymerase chain reaction, RT-PCR）中并行生成的多组扩增子开展下一代测序（next generation sequencing, NGS），并采用二维唯一双索引策略，总计使用4个索引，可实现测序读段（reads）与单个样本的精准且可扩展的对应分配。研究团队通过合成RNA与病毒粒子的稀释液对该方法进行校准，证明其可实现低至数个分子的检测灵敏度；并将其应用于数百份患者样本，验证了该方法在多种样本类型下均具备稳定的检测性能。在临床场景与人类诊断实验室中，以金标准定量逆转录聚合酶链式反应（quantitative RT-PCR, qRT-PCR）为参照开展的双盲基准测试结果显示，该方法在循环阈值（Cycle threshold, Ct）达36时仍可保持稳定的检测性能。经估算，该方法的假阳性率为0.04%~0.1%，该误差大概率源于样本移液过程中的交叉污染。除SARS-CoV2外，SARSeq还可检测甲型流感病毒、乙型流感病毒以及人鼻病毒，且可便捷扩展以实现其他病原体的检测。综上，SARSeq是可满足大流行期间规模需求的呼吸道疾病鉴别诊断理想平台。基于RNA测序的唾液分析方法SARSeq可实现大规模并行且兼具成本效益的SARS-CoV2检测。本研究纳入的患者样本为剩余样本。[贡献者] 维也纳新冠病毒检测倡议工作组全体成员