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

TLDR

Viral whole‑genome sequencing provides critical insight into SARS‑CoV‑2 transmission and evolution, and Oxford Nanopore Technologies long‑read devices promise faster, cheaper, and more portable sequencing, but adoption has been limited by accuracy concerns. The study aims to rigorously evaluate the analytical performance of Oxford Nanopore Technologies sequencing for SARS‑CoV‑2 by comparing it with Illumina on 157 matched patient specimens and synthetic controls. The authors performed whole‑genome sequencing on 157 matched SARS‑CoV‑2–positive patient samples and synthetic RNA controls using both ONT and Illumina platforms. Despite ONT’s higher error rates, consensus‑level accuracy exceeded 99 % for SNV detection at ≥60× coverage, and ONT revealed diverse structural variants corroborated by Illumina, though it failed to detect short indels and low‑frequency variants, demonstrating suitability for SARS‑CoV‑2 genome analysis and supporting broader adoption.

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.