tetano
Editor, Senior Moderator
Nat Commun
. 2020 Dec 9;11(1):6272.
doi: 10.1038/s41467-020-20075-6.
Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis
Rowena A Bull[SUP] 1 2 [/SUP], Thiruni N Adikari[SUP] 1 2 [/SUP], James M Ferguson[SUP] 3 [/SUP], Jillian M Hammond[SUP] 3 [/SUP], Igor Stevanovski[SUP] 3 [/SUP], Alicia G Beukers[SUP] 4 [/SUP], Zin Naing[SUP] 2 5 [/SUP], Malinna Yeang[SUP] 2 5 [/SUP], Andrey Verich[SUP] 1 [/SUP], Hasindu Gamaarachchi[SUP] 3 6 [/SUP], Ki Wook Kim[SUP] 5 7 [/SUP], Fabio Luciani[SUP] 1 2 [/SUP], Sacha Stelzer-Braid[SUP] 2 5 [/SUP], John-Sebastian Eden[SUP] 8 9 [/SUP], William D Rawlinson[SUP] 2 5 7 10 [/SUP], Sebastiaan J van Hal[SUP] 4 11 [/SUP], Ira W Deveson[SUP] 12 13 [/SUP]
Affiliations
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.
. 2020 Dec 9;11(1):6272.
doi: 10.1038/s41467-020-20075-6.
Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis
Rowena A Bull[SUP] 1 2 [/SUP], Thiruni N Adikari[SUP] 1 2 [/SUP], James M Ferguson[SUP] 3 [/SUP], Jillian M Hammond[SUP] 3 [/SUP], Igor Stevanovski[SUP] 3 [/SUP], Alicia G Beukers[SUP] 4 [/SUP], Zin Naing[SUP] 2 5 [/SUP], Malinna Yeang[SUP] 2 5 [/SUP], Andrey Verich[SUP] 1 [/SUP], Hasindu Gamaarachchi[SUP] 3 6 [/SUP], Ki Wook Kim[SUP] 5 7 [/SUP], Fabio Luciani[SUP] 1 2 [/SUP], Sacha Stelzer-Braid[SUP] 2 5 [/SUP], John-Sebastian Eden[SUP] 8 9 [/SUP], William D Rawlinson[SUP] 2 5 7 10 [/SUP], Sebastiaan J van Hal[SUP] 4 11 [/SUP], Ira W Deveson[SUP] 12 13 [/SUP]
Affiliations
- PMID: 33298935
- DOI: 10.1038/s41467-020-20075-6
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.