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

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, 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 >98% 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.

High Throughput Nanopore Sequencing of SARS-CoV-2 Viral Genomes from Patient Samples

“…Nanopolish uses fast5 signal data while the other, Medaka, does not. In our experience both workflows tend to give consistent results with high variant and SNV detection accuracy (13). We have preferentially used Medaka due to its speed and GPU compatibility.…”

RAY: CRISPR diagnostic for rapid and accurate detection of SARS-CoV2 variants on a paper strip

“…The main advantage of sequencing COVID-19 samples is the generation of cumulative information about all mutations co-existing in a given variant 44,,45 . This information is valuable to track and trace how lineages evolve and has led to the identification of mutations in the first place.…”

“…Sequencing generates a large amount of data and requires physical storage and high-performance computing methodologies to record, analyze and interpret the data 44,45 . In addition, dedicated personnel with a significant understanding of sequence analysis and programming skills are necessary for identifying variants within reads generated from a sample.…”

Genomic epidemiology of COVID-19 in care homes in the east of England

“…If an individual is infected with more than one clone at significant frequency, it is also possible for stochastic variation in read counts for the two variants to yield different consensus calls at the variant locus. However, larger studies have systematically evaluated sequencing quality for SARS-CoV-2 between Oxford Nanopore Technology (ONT) and Illumina, and demonstrated highly accurate consensus-level sequence determination ( Bull et al, 2020 ). Given this degree of consensus sequence accuracy, and because transcluster uses a transmission probability cut-off based on integrating pairwise SNP and temporal differences (rather than relying solely on a strict SNP cut-off), limited sequencing noise is unlikely to have a substantial impact on the clusters identified.…”