Dataset Information

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore.

ABSTRACT: Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing ( https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w ) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an updated primer scheme with 22 additional primers to improve genome coverage, ii) a streamlined library preparation workflow which improves demultiplexing rate for up to 96 samples and reduces hands-on time by several hours and iii) cost savings which bring the reagent cost down to £10 per sample making it practical for individual labs to sequence thousands of SARS-CoV-2 genomes to support national and international genomic epidemiology efforts.

SUBMITTER: Tyson JR

PROVIDER: S-EPMC7480024 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore.

Tyson John R JR James Phillip P Stoddart David D Sparks Natalie N Wickenhagen Arthur A Hall Grant G Choi Ji Hyun JH Lapointe Hope H Kamelian Kimia K Smith Andrew D AD Prystajecky Natalie N Goodfellow Ian I Wilson Sam J SJ Harrigan Richard R Snutch Terrance P TP Loman Nicholas J NJ Quick Joshua J

bioRxiv : the preprint server for biology 20200904

Genome sequencing has been widely deployed to study the evolution of SARS-CoV-2 with more than 90,000 genome sequences uploaded to the GISAID database. We published a method for SARS-CoV-2 genome sequencing (https://www.protocols.io/view/ncov-2019-sequencing-protocol-bbmuik6w) online on January 22, 2020. This approach has rapidly become the most popular method for sequencing SARS-CoV-2 due to its simplicity and cost-effectiveness. Here we present improvements to the original protocol: i) an upda ...[more]

PMID: 32908977

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Project description:BACKGROUND:Dengue virus (DENV) infects hundreds of thousands of people annually in Indonesia. However, DENV sequence data from the country are limited, as samples from outbreaks must be shipped across long-distances to suitably equipped laboratories to be sequenced. This approach is time-consuming, expensive, and frequently results in failure due to low viral load or degradation of the RNA genome. METHODS:We evaluated a method designed to address this challenge, using the 'Primal Scheme' multiplex PCR tiling approach to rapidly generate short, overlapping amplicons covering the complete DENV coding-region, and sequencing the amplicons on the portable Nanopore MinION device. The resulting sequence data was assessed in terms of genome coverage, consensus sequence accuracy and by phylogenetic analysis. RESULTS:The multiplex approach proved capable of producing near complete coding-region coverage from all samples tested ([Formula: see text] = 99.96%, n?=?18), 61% of which could not be fully amplified using the current, long-amplicon PCR, approach. Nanopore-generated consensus sequences were found to be between 99.17-99.92% identical to those produced by high-coverage Illumina sequencing. Consensus accuracy could be improved by masking regions below 20X coverage depth (99.69-99.92%). However, coding-region coverage was reduced at this depth ([Formula: see text] = 93.48%). Nanopore and Illumina consensus sequences generated from the same samples formed monophyletic clades on phylogenetic analysis, and Indonesian consensus sequences accurately clustered by geographical origin. CONCLUSION:The multiplex, short-amplicon approach proved superior for amplifying DENV genomes from clinical samples, particularly when the virus was present at low concentrations. The accuracy of Nanopore-generated consensus sequences from these amplicons was sufficient for identifying the geographic origin of the samples, demonstrating that the approach can be a useful tool for identifying and monitoring DENV clades circulating in low-resource settings across Indonesia. However, the inaccuracies in Nanopore-generated consensus sequences mean that the approach may not be appropriate for higher resolution transmission studies, particularly when more accurate sequencing technologies are available.

Dataset Information

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore.

Publications

Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore.

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