The emergence of SARS-CoV-2 has caused the current COVID-19 pandemic with catastrophic societal impact. Because many individuals shed virus for days before symptom onset, and many show mild or no symptoms, an emergent and unprecedented need exists for development and deployment of sensitive and high throughput molecular diagnostic tests. RNA-mediated oligonucleotide Annealing Selection and Ligation with next generation DNA sequencing (RASL-seq) is a highly multiplexed technology for targeted analysis of polyadenylated mRNA, which incorporates sample barcoding for massively parallel analyses. Here we present a more generalized method, capture RASL-seq ("cRASL-seq"), which enables analysis of any targeted pathogen- (and/or host-) associated RNA molecules. cRASL-seq enables highly sensitive (down to ~1-100 pfu/ml or cfu/ml) and highly multiplexed (up to ~10,000 target sequences) detection of pathogens. Importantly, cRASL-seq analysis of COVID-19 patient nasopharyngeal (NP) swab specimens does not involve nucleic acid extraction or reverse transcription, steps that have caused testing bottlenecks associated with other assays. Our simplified workflow additionally enables the direct and efficient genotyping of selected, informative SARS-CoV-2 polymorphisms across the entire genome, which can be used for enhanced characterization of transmission chains at population scale and detection of viral clades with higher or lower virulence. Given its extremely low per-sample cost, simple and automatable protocol and analytics, probe panel modularity, and massive scalability, we propose that cRASL-seq testing is a powerful new surveillance technology with the potential to help mitigate the current pandemic and prevent similar public health crises.

中文翻译：

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高度多路复用的寡核苷酸探针连接测试可实现高效的无提取 SARS-CoV-2 检测和病毒基因分型。

SARS-CoV-2 的出现导致了当前的 COVID-19 大流行，带来了灾难性的社会影响。由于许多人在症状出现前几天就已经排出病毒，并且许多人表现出轻微或没有症状，因此迫切需要开发和部署灵敏和高通量的分子诊断测试。RNA 介导的寡核苷酸退火选择和连接与下一代 DNA 测序 (RASL-seq) 是一种高度多路复用的技术，用于多腺苷酸化 mRNA 的靶向分析，其中包含用于大规模并行分析的样本条形码。在这里，我们提出了一种更通用的方法，捕获 RASL-seq（“cRASL-seq”），它可以分析任何目标病原体（和/或宿主）相关的 RNA 分子。cRASL-seq 可实现高度灵敏（低至 ~1-100 pfu/ml 或 cfu/ml）和高度多重（高达 ~10,000 个目标序列）的病原体检测。重要的是，对 COVID-19 患者鼻咽 (NP) 拭子标本的 cRASL-seq 分析不涉及核酸提取或逆转录，这些步骤已导致与其他检测相关的测试瓶颈。此外，我们简化的工作流程还可以对整个基因组中选定的、信息丰富的 SARS-CoV-2 多态性进行直接有效的基因分型，这可用于增强群体规模传播链的表征以及检测具有较高或较低毒力的病毒进化枝。鉴于其极低的每个样本成本、简单且可自动化的协议和分析、探针板模块化以及大规模的可扩展性，