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

Nat Commun. 2020 Dec 9;11(1):6272. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • COVID-19 / diagnosis
  • COVID-19 / virology
  • Genome, Viral
  • Humans
  • Nanopore Sequencing / methods*
  • RNA, Viral
  • SARS-CoV-2* / genetics
  • SARS-CoV-2* / isolation & purification
  • Sensitivity and Specificity
  • Whole Genome Sequencing / methods*

Substances

  • RNA, Viral