COVID-19(2019年冠状病毒病)在全球范围内造成了一场重大流行病;然而,关于该病毒的流行病学和进化尚未有很多了解,部分原因是报道的全长SARS-CoV-2(严重急性呼吸道综合征冠状病毒2)基因组的缺乏.原因之一是直接从临床样本中对SARS-CoV-2进行测序所面临的挑战尚未完全解决,即,具有低病毒载量的测序样品通常导致用于分析的病毒读数不足。
我们应用了一种新的基于多重PCR扩增子(扩增子)和基于杂交捕获(捕获)的测序,以及在检索完整基因组中的超高通量meta基因组(meta)测序,SARS-CoV-2的个体间和个体内变异来自培养分离株的血清稀释液,和八个临床样本,涵盖一系列样本类型和病毒载量。我们还检查并比较了灵敏度,准确度,以及这些方法的其他特征。
我们证明了扩增子和捕获方法都有效地从临床样品中富集SARS-CoV-2含量,而扩增子的富集效率超过了更具挑战性的样品中的捕获效率。我们发现捕获在识别样本间变异方面不如meta和扩增子准确,而扩增子方法在研究样品内变化方面不如其他两种方法准确,表明扩增子测序不适合研究严重依赖宿主内动力学的病毒-宿主相互作用和病毒传播。我们说明了meta在SARS-CoV-2之外的临床样本中揭示了丰富的遗传信息,为临床诊断和治疗提供了参考。考虑到上述所有因素和成本效益,我们对SARS-CoV-2在不同情况下如何选择测序策略提出了指导意见。
这是,据我们所知,第一项工作是使用基于扩增和捕获的全基因组测序系统地研究SARS-CoV-2的个体间和个体内变异,以及多种方法之间的第一个比较研究。我们的工作为SARS-CoV-2和其他新兴病毒的基因组测序和分析提供了实用的解决方案。
COVID-19 (coronavirus disease 2019) has caused a major epidemic worldwide; however, much is yet to be known about the epidemiology and evolution of the virus partly due to the scarcity of full-length SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genomes reported. One reason is that the challenges underneath sequencing SARS-CoV-2 directly from clinical samples have not been completely tackled, i.e., sequencing samples with low viral load often results in insufficient viral reads for analyses.
We applied a novel multiplex PCR amplicon (amplicon)-based and hybrid capture (capture)-based sequencing, as well as ultra-high-throughput metatranscriptomic (meta) sequencing in retrieving complete genomes, inter-individual and intra-individual variations of SARS-CoV-2 from serials dilutions of a cultured isolate, and eight clinical samples covering a range of sample types and viral loads. We also examined and compared the sensitivity, accuracy, and other characteristics of these approaches in a comprehensive manner.
We demonstrated that both amplicon and capture methods efficiently enriched SARS-CoV-2 content from clinical samples, while the enrichment efficiency of amplicon outran that of capture in more challenging samples. We found that capture was not as accurate as meta and amplicon in identifying between-sample variations, whereas amplicon method was not as accurate as the other two in investigating within-sample variations, suggesting amplicon sequencing was not suitable for studying virus-host interactions and viral transmission that heavily rely on intra-host dynamics. We illustrated that meta uncovered rich genetic information in the clinical samples besides SARS-CoV-2, providing references for clinical diagnostics and therapeutics. Taken all factors above and cost-effectiveness into consideration, we proposed guidance for how to choose sequencing strategy for SARS-CoV-2 under different situations.
This is, to the best of our knowledge, the first work systematically investigating inter- and intra-individual variations of SARS-CoV-2 using amplicon- and capture-based whole-genome sequencing, as well as the first comparative study among multiple approaches. Our work offers practical solutions for genome sequencing and analyses of SARS-CoV-2 and other emerging viruses.