PT  - JOURNAL ARTICLE
AU  - Rowena A. Bull
AU  - Thiruni Adikari
AU  - James M. Ferguson
AU  - Jillian M. Hammond
AU  - Igor Stevanovski
AU  - Alicia G. Beukers
AU  - Zin Naing
AU  - Malinna Yeang
AU  - Andrey Verich
AU  - Hasindu Gamaarachchi
AU  - Ki Wook Kim
AU  - Fabio Luciani
AU  - Sacha Stelzer-Braid
AU  - John-Sebastian Eden
AU  - William D. Rawlinson
AU  - Sebastiaan J. van Hal
AU  - Ira W. Deveson
TI  - Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis
AID  - 10.1101/2020.08.04.236893
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.08.04.236893
4099  - http://biorxiv.org/content/early/2020/10/20/2020.08.04.236893.short
4100  - http://biorxiv.org/content/early/2020/10/20/2020.08.04.236893.full
AB  - 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.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.