Most severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostic tests have relied on RNA extraction followed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays. Whereas automation improved logistics and different pooling strategies increased testing capacity, highly multiplexed next-generation sequencing (NGS) diagnostics remain a largely untapped resource. NGS tests have the potential to markedly increase throughput while providing crucial SARS-CoV-2 variant information. Current NGS-based detection and genotyping assays for SARS-CoV-2 are costly, mostly due to parallel sample processing through multiple steps. Here, we have established ApharSeq, in which samples are barcoded in the lysis buffer and pooled before reverse transcription. We validated this assay by applying ApharSeq to more than 500 clinical samples from the Clinical Virology Laboratory at Hadassah hospital in a robotic workflow. The assay was linear across five orders of magnitude, and the limit of detection was Ct 33 (~1000 copies/ml, 95% sensitivity) with >99.5% specificity. ApharSeq provided targeted high-confidence genotype information due to unique molecular identifiers incorporated into this method. Because of early pooling, we were able to estimate a 10- to 100-fold reduction in labor, automated liquid handling, and reagent requirements in high-throughput settings compared to current testing methods. The protocol can be tailored to assay other host or pathogen RNA targets simultaneously. These results suggest that ApharSeq can be a promising tool for current and future mass diagnostic challenges.

中文翻译：

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早期样本标记和汇集可同时进行 SARS-CoV-2 检测和变异测序

大多数严重急性呼吸系统综合症冠状病毒 2 (SARS-CoV-2) 诊断测试依赖于 RNA 提取，然后进行逆转录定量聚合酶链反应 (RT-qPCR) 测定。尽管自动化改进了物流并且不同的合并策略提高了测试能力，但高度多重的下一代测序 (NGS) 诊断仍然是一个很大程度上未开发的资源。NGS 测试有可能显着提高通量，同时提供关键的 SARS-CoV-2 变异信息。当前基于 NGS 的 SARS-CoV-2 检测和基因分型分析成本高昂，这主要是由于通过多个步骤进行的并行样本处理。在这里，我们建立了 ApharSeq，其中样本在裂解缓冲液中进行条形码标记，并在逆转录前汇集。我们通过在机器人工作流程中将 ApharSeq 应用于来自 Hadassah 医院临床病毒学实验室的 500 多个临床样本来验证该测定。该测定在五个数量级内呈线性，检测限为 Ct 33（~1000 拷贝/毫升，95% 的灵敏度），特异性 > 99.5%。由于该方法中包含独特的分子标识符，ApharSeq 提供了有针对性的高可信度基因型信息。由于早期汇集，我们能够估计与当前测试方法相比，高通量设置中的劳动力、自动液体处理和试剂需求减少了 10 到 100 倍。该协议可以定制以同时检测其他宿主或病原体 RNA 目标。这些结果表明 ApharSeq 可以成为应对当前和未来大规模诊断挑战的有前途的工具。