Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis
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 >98% 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.Competing Interest StatementJ.F. & H.G. have previously received travel and accommodation expenses to attend ONT conferences. The authors declare no other competing financial interests.
|Authors||Bull, Rowena A.; Adikari, Thiruni; Hammond, Jillian M.; Stevanovski, Igor; Ferguson, James M.; Beukers, Alicia G.; Naing, Zin; Yeang, Malinna; Verich, Andrey; Gamaarachichi, Hasindu; Kim, Ki Wook; Luciani, Fabio; Stelzer-Braid, Sacha; Eden, John-Sebastian; Rawlinson, William D.; van Hal, Sebastiaan J.; Deveson, Ira W.|
|Responsible Garvan Author|
|Publisher Name||Nature Communications|
|URL link to publisher's version||https://pubmed.ncbi.nlm.nih.gov/33298935/|