Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
US Army Medical Research Institute of Infectious Disease Center for Genome Sciences, Fort Detrick, Maryland, USA.
J Clin Microbiol. 2021 Apr 20;59(5). doi: 10.1128/JCM.02784-20.
The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here, we report a simple and efficient workflow for whole-genome sequencing utilizing one-step reverse transcription-PCR (RT-PCR) amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm integrated fluidic circuit (IFC) and instruments to amplify 48 samples with 39 pairs of primers, including 35 custom-designed primer pairs and four additional primer pairs from the ARTIC network protocol v3. Application of this method on RNA samples from both viral isolates and clinical specimens demonstrates robustness and efficiency in obtaining the full genome sequence of SARS-CoV-2.
长期肆虐的全球 COVID-19 大流行要求及时对 SARS-CoV-2 病毒进行基因组研究。在这里，我们报告了一种简单而高效的全基因组测序工作流程，该流程利用微流控平台上的一步法逆转录-PCR（RT-PCR）扩增，随后进行 MiSeq 扩增子测序。该方法使用 Fluidigm 集成式流体回路（IFC）和仪器对 48 个样本进行扩增，使用 39 对引物，包括 35 对定制设计的引物对和来自 ARTIC 网络协议 v3 的另外 4 对引物。该方法在病毒分离株和临床标本的 RNA 样本上的应用证明了其获得 SARS-CoV-2 全长基因组序列的稳健性和高效性。
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