Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis

Rowena A. Bull, Thiruni N. Adikari, James M. Ferguson, Jillian M. Hammond, Igor Stevanovski, Alicia G. Beukers, Zin Naing, Malinna Yeang, Andrey Verich, Hasindu Gamaarachchi, Ki Wook Kim, Fabio Luciani, Sacha Stelzer-Braid, John-Sebastian Eden, William D. Rawlinson, Sebastiaan J. Hal and Ira W. Deveson ()
Additional contact information
Rowena A. Bull: The Kirby Institute for Infection and Immunity, University of New South Wales
Thiruni N. Adikari: The Kirby Institute for Infection and Immunity, University of New South Wales
James M. Ferguson: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research
Jillian M. Hammond: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research
Igor Stevanovski: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research
Alicia G. Beukers: Royal Prince Alfred Hospital
Zin Naing: School of Medical Sciences, Faculty of Medicine, University of New South Wales
Malinna Yeang: School of Medical Sciences, Faculty of Medicine, University of New South Wales
Andrey Verich: The Kirby Institute for Infection and Immunity, University of New South Wales
Hasindu Gamaarachchi: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research
Ki Wook Kim: Virology Research Laboratory, Serology and Virology Division (SAViD), NSW Health Pathology, Prince of Wales Hospital
Fabio Luciani: The Kirby Institute for Infection and Immunity, University of New South Wales
Sacha Stelzer-Braid: School of Medical Sciences, Faculty of Medicine, University of New South Wales
John-Sebastian Eden: Marie Bashir Institute for Infectious Diseases and Biosecurity & Sydney Medical School, The University of Sydney
William D. Rawlinson: School of Medical Sciences, Faculty of Medicine, University of New South Wales
Sebastiaan J. Hal: Royal Prince Alfred Hospital
Ira W. Deveson: Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research

Nature Communications, 2020, vol. 11, issue 1, 1-8

Abstract: Abstract 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.

Date: 2020
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DOI: 10.1038/s41467-020-20075-6

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