Monitoring new mutations in SARS-CoV-2 provides crucial information for identifying diagnostic and therapeutic targets and important insights to achieve a more effective COVID-19 control strategy. Next generation sequencing (NGS) technologies have been widely used for whole genome sequencing (WGS) of SARS-CoV-2. While various NGS methods have been reported, one chief limitation has been the complexity of the workflow, limiting the scalability. Here, we overcome this limitation by designing a laboratory workflow optimized for high-throughput studies. The workflow utilizes modified ARTIC network v3 primers for SARS-CoV-2 whole genome amplification. NGS libraries were prepared by a 2-step PCR method, similar to a previously reported tailed PCR method, with further optimizations to improve amplicon balance, to minimize amplicon dropout for viral genomes harboring primer-binding site mutation(s), and to integrate robotic liquid handlers. Validation studies demonstrated that the optimized workflow can process up to 2688 samples in a single sequencing run without compromising sensitivity and accuracy and with fewer amplicon dropout events compared to the standard ARTIC protocol. We additionally report results for over 65,000 SARS-CoV-2 whole genome sequences from clinical specimens collected in the United States between January and September of 2021, as part of an ongoing national genomics surveillance effort.
【저자키워드】 Infectious-disease diagnostics, Whole genome amplification, 【초록키워드】 COVID-19, SARS-CoV-2, Mutation, Sequencing, NGS, diagnostic, Laboratory, amplification, sensitivity, Whole genome sequencing, validation, Viral, Surveillance, Accuracy, viral genomes, monitoring, United States, WGS, information, viral genome, ARTIC protocol, therapeutic target, balance, complexity, specimen, scalability, effort, primer, National, whole genome, while, whole genome sequence, PCR method, effective, event, IMPROVE, collected, reported, the United State, provide, overcome, demonstrated, 【제목키워드】 SARS-CoV-2, Sequencing, Laboratory, development, whole genome,