The Kirby Institute for Infection and Immunity, University of New South Wales, Sydney, NSW, Australia.
School of Medical Sciences, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.
Nat Commun. 2020 Dec 9;11(1):6272. doi: 10.1038/s41467-020-20075-6.
Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.
病毒全基因组测序（WGS）为严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的传播和进化提供了重要的见解。与 SARS-CoV-2 病毒 WGS 的既定短读测序平台（例如 Illumina）相比，牛津纳米孔技术（ONT）的长读测序设备在周转时间、便携性和成本方面具有显著优势。然而，由于测序准确性方面的常见问题，ONT 测序在 SARS-CoV-2 监测中的应用受到限制。为了解决这个问题，我们在这里使用 ONT 和 Illumina 平台对 157 个匹配的 SARS-CoV-2 阳性患者样本和合成 RNA 对照进行病毒 WGS，从而对分析性能进行了严格评估。我们报告说，尽管在 ONT 测序reads 中观察到了升高的错误率，但仍实现了高度准确的共识水平序列确定，单核苷酸变异（SNV）的检测灵敏度>99%，在最低约 60 倍覆盖深度以上的精度>99%，从而确保了 SARS-CoV-2 基因组分析的适用性。ONT 测序还在 SARS-CoV-2 样本中鉴定了令人惊讶的结构变异多样性，这些多样性得到了来自匹配样本的短读测序的证据支持。然而，ONT 测序未能准确检测到短插入缺失和低频读数变异。这项对 SARS-CoV-2 WGS 分析性能的系统评估将促进 ONT 测序在本地、国家和国际 COVID-19 公共卫生计划中的广泛采用。
