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, we performed 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. 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.

中文翻译：

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纳米孔测序对SARS-CoV-2基因组快速分析的分析有效性

病毒全基因组测序（WGS）提供了对严重急性呼吸系统综合症冠状病毒2（SARS-CoV-2）的传播和进化的重要见解。与已建立的病毒WGS的短读测序平台（例如Illumina）相比，Oxford Nanopore Technologies（ONT）的长读测序设备有望在周转时间，便携性和成本方面取得显着改善。但是，由于对测序准确性的普遍关注，在SARS-CoV-2监视中采用ONT测序受到了限制。为了解决这个问题，我们在157个匹配的SARS-CoV-2阳性患者标本和合成RNA对照样品上使用ONT和Illumina平台进行了病毒WGS，可对分析性能进行严格评估。尽管在ONT测序读取中观察到错误率升高，达到了高度准确的共有水平序列测定，检测到的单核苷酸变体（SNV）在高于最小〜60倍覆盖深度的情况下，以> 99％的灵敏度和> 99％的精度检测，从而确保了对SARS-CoV-2基因组分析的适用性。ONT测序还发现了SARS-CoV-2标本中令人惊讶的结构变异多样性，这得益于对匹配样品进行短读测序的证据。但是，ONT测序无法在低读取次数频率下准确检测到较短的indel和变异。对SARS-CoV-2 WGS的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。在最低约60倍的覆盖深度之上具有99％的精度，从而确保了对SARS-CoV-2基因组分析的适用性。ONT测序还发现了SARS-CoV-2标本中令人惊讶的结构变异多样性，这得益于对匹配样品进行短读测序的证据。但是，ONT测序无法在低读取次数频率下准确检测到较短的indel和变异。对SARS-CoV-2 WGS的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。在最小约60倍的覆盖深度之上，具有99％的精度，从而确保了对SARS-CoV-2基因组分析的适用性。ONT测序还发现了SARS-CoV-2标本中令人惊讶的结构变异多样性，这得益于对匹配样品进行短读测序的证据。但是，ONT测序无法在低读取次数频率下准确检测到短插入缺失和变异。对SARS-CoV-2 WGS的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。ONT测序还发现了SARS-CoV-2标本中令人惊讶的结构变异多样性，这得益于对匹配样品进行短读测序的证据。但是，ONT测序无法在低读取次数频率下准确检测到较短的indel和变异。对SARS-CoV-2 WGS的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。ONT测序还发现了SARS-CoV-2标本中令人惊讶的结构变异多样性，这得益于对匹配样品进行短读测序的证据。但是，ONT测序无法在低读取次数频率下准确检测到短插入缺失和变异。对SARS-CoV-2 WGS的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。