Rapid Acquisition of High-Quality SARS-CoV-2 Genome via Amplicon-Oxford Nanopore Sequencing

Abstract

Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary tracing. While the rapid acquisition of SARS-CoV-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid background. To address this issue, we modified and evaluated an approach by utilizing SARS-CoV-2-specific amplicon amplification and Oxford Nanopore PromethION platform. This workflow started with the throat swab of the COVID-19 patient, combined reverse transcript PCR, and multi-amplification in one-step to shorten the experiment time, then can quickly and steadily obtain high-quality SARS-CoV-2 genome within 24 h. A comprehensive evaluation of the method was conducted in 42 samples: the sequencing quality of the method was correlated well with the viral load of the samples; high-quality SARS-CoV-2 genome could be obtained stably in the samples with Ct value up to 39.14; data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20; variation analysis indicated that the method can detect the existing and emerging genomic mutations as well; Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of Nanopore sequencing, making it as low as 0.4/10,000 bp. In summary, high-quality SARS-CoV-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARS-CoV-2. Supplementary Information The online version contains supplementary material available at 10.1007/s12250-021-00378-8.

poreCov-An Easy to Use, Fast, and Robust Workflow for SARS-CoV-2 Genome Reconstruction via Nanopore Sequencing

“…Illumina is still the most dominant approach for SARS-CoV-2 sequencing and genome reconstruction ( Table 1 ). However, nanopore sequencing via tiled amplicon-based genome reconstruction ( Quick et al, 2017 ; Yan et al, 2021 ) is also heavily used and of particular interest, as laboratories can enter sequencing with a comparably low initial investment. While many laboratories can implement nanopore sequencing techniques rapidly, bioinformatics know-how, on the other hand, is still very scarce in science ( Hodcroft et al, 2021 ).…”

Developing an Amplification Refractory Mutation System-Quantitative Reverse Transcription-PCR Assay for Rapid and Sensitive Screening of SARS-CoV-2 Variants of Concern

“…3 ). The 88 samples included 40 SARS-CoV-2-positive samples previously collected in January 2020 and sequenced by our laboratory ( 15 ) and 48 samples collected from suspected COVID-19 international travelers at the Wuhan international airport from May to July 2021. Among these specimens, 9 samples were found PCR negative for SARS-CoV-2 and 79 samples were found positive using commercial SARS-CoV-2 RT-PCR kits.…”

“…Next-generation sequencing (NGS) is the most accurate and widely used method for identification of SARS-CoV-2 VOCs since genome-wide mutations can be shown ( 15 , 16 ). However, NGS represents a significant constraint for most resource-limited laboratories.…”

Management of a large outbreak of COVID-19 at a British Army training centre: lessons for the future

“…Sequencing was performed using the ARTIC nCOV-2019 sequencing protocol V.3 (LoCost), using primers from Integrated DNA Technologies (Iowa, USA). 4 5 Samples were sequenced on a GridION X5 system (Oxford Nanopore Technologies, UK) along with a synthetic positive control (Twist Biosciences, USA) and a nuclease-free water negative control. Analysis of the resulting data was performed using the ARTIC fieldbioinformatics toolkit V.1.2.1 ( https://github.com/artic-network/artic-ncov2019 ).…”