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. Nanopore sequencing (ONT) has been used in SARS-CoV-2 studies, however adoption of ONT for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. Here, the authors perform a comprehensive evaluation of ONT analytical performance on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls.
【저자키워드】 Next-generation sequencing, Viral genetics, 【초록키워드】 COVID-19, whole-genome sequencing, public health, Evolution, SARS-CoV-2, Sequencing, variant, Local, Transmission, coronavirus 2, RNA, improvement, sensitivity, Coverage, Accuracy, SARS-CoV-2 genome, International, Patient, nanopore sequencing, respiratory, WGS, Illumina, Critical, platform, Single nucleotide variant, Evidence, Analysis, SARS-CoV-2 surveillance, Structural variation, Precision, frequencies, sequence, specimen, error rate, National, INDEL, SNVs, sequencing reads, widespread, controls, detect, elevated, facilitate, supported, provide, ONT, Oxford Nanopore Technology, short-read sequencing, 【제목키워드】 Nanopore, Sequencing, SARS-CoV-2 genome, Validity, Analysis,