Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

Freed, Nikki E.; Vlková, Markéta; Faisal, Muhammad B.; Silander, Olin K.

doi:10.1093/biomethods/bpaa014

Cited by 232 publications

(202 citation statements)

References 9 publications

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“…In brief, viral extracts were prepared from respiratory tract samples in which SARS-CoV-2 was detected by rRT-PCR by using World Health Organization–recommended primers and probes targeting the envelope and nucleocapsid genes. Extracted RNA from SARS-CoV-2–positive samples was subjected to whole-genome sequencing by following the ARTIC network protocol version 3 ( https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye ) and using the Massey University 1200-bp primer set ( https://www.protocols.io/view/ncov-2019-sequencing-protocol-rapid-barcoding-1200-bh7hj9j6 ) ( 16 ).…”

Section: Methodsmentioning

confidence: 99%

Use of Genomics to Track Coronavirus Disease Outbreaks, New Zealand

Geoghegan¹,

Douglas²,

Ren³

et al. 2021

Emerg. Infect. Dis.

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Section: Methodsmentioning

confidence: 99%

Use of Genomics to Track Coronavirus Disease Outbreaks, New Zealand

Geoghegan¹,

Douglas²,

Ren³

et al. 2021

Emerg. Infect. Dis.

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“…The SARS-CoV-2 amplicons were generated according to the protocol described by Freed et al [29] however the described primer set was replaced with those from the ArticV3 protocol [30], generating a series of 400-bp in lieu of 1200-bp amplicons and with additional modifications. Briefly, 100 uM stock primer pools were prepared for each set of primers (i.e., odd and even) by combining equal volumes of the appropriate primers (LabReady, 100 uM IDTE, pH 8.0; IDT) and then diluted to 10 uM prior to use.…”

Section: Methodsmentioning

confidence: 99%

Metagenomic sequencing of municipal wastewater provides a near-complete SARS-CoV-2 genome sequence identified as the B.1.1.7 variant of concern from a Canadian municipality concurrent with an outbreak

Landgraff

Wang

Buchanan

et al. 2021

Preprint

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Laboratory-based wastewater surveillance for SARS-CoV-2, the causative agent of the ongoing COVD-19 pandemic, can be conducted using RT-qPCR-based screening of municipal wastewater samples. Although it provides rapid viral detection and can inform SARS-CoV-2 abundance in wastewater, this approach lacks the resolution required for viral genotyping and does not support tracking of viral genome evolution. The recent emergence of several variants of concern, a result of mutations across the genome including the accrual of important mutations within the viral spike glycoprotein, has highlighted the need for a method capable of detecting the cohort of mutations associated with these and newly emerging genotypes. Here we provide an innovative methodology for the recovery of a near-complete SARS-CoV-2 sequence from a wastewater sample collected from across Canadian municipalities including one that experienced a significant outbreak attributable to the SARS-CoV-2 B.1.1.7 variant of concern. Our results demonstrate that a combined interrogation of genome consensus-level sequences and alternative alleles enables the identification of a SARS-CoV-2 variant of concern and the detection of a new allele within a viral accessory gene that may be representative of a recently evolved B.1.1.7 sublineage.

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“…WGS offers increased resolution at multiple epidemiological scales, from investigating global virus traffic networks to elucidating individual transmission events within outbreaks [ 15 , 16 ]. The recent SARS-CoV-2 epidemic has highlighted the utility of amplicon-based WGS methods as a cost-effective, rapid method to sequence the whole-genome approach [ 17 , 18 , 19 ]. The purpose of this study was to develop a simple and robust amplicon-based method for sequencing the HMPV full-length genome with the aim to inform a better understanding of its molecular epidemiology.…”

Section: Introductionmentioning

confidence: 99%

An Amplicon-Based Approach for the Whole-Genome Sequencing of Human Metapneumovirus

Tulloch

Kok

Carter

et al. 2021

Viruses

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Human metapneumovirus (HMPV) is an important cause of upper and lower respiratory tract disease in individuals of all ages. It is estimated that most individuals will be infected by HMPV by the age of five years old. Despite this burden of disease, there remain caveats in our knowledge of global genetic diversity due to a lack of HMPV sequencing, particularly at the whole-genome scale. The purpose of this study was to create a simple and robust approach for HMPV whole-genome sequencing to be used for genomic epidemiological studies. To design our assay, all available HMPV full-length genome sequences were downloaded from the National Center for Biotechnology Information (NCBI) GenBank database and used to design four primer sets to amplify long, overlapping amplicons spanning the viral genome and, importantly, specific to all known HMPV subtypes. These amplicons were then pooled and sequenced on an Illumina iSeq 100 (Illumina, San Diego, CA, USA); however, the approach is suitable to other common sequencing platforms. We demonstrate the utility of this method using a representative subset of clinical samples and examine these sequences using a phylogenetic approach. Here we present an amplicon-based method for the whole-genome sequencing of HMPV from clinical extracts that can be used to better inform genomic studies of HMPV epidemiology and evolution.

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Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding

Cited by 232 publications

References 9 publications

Use of Genomics to Track Coronavirus Disease Outbreaks, New Zealand

Use of Genomics to Track Coronavirus Disease Outbreaks, New Zealand

Metagenomic sequencing of municipal wastewater provides a near-complete SARS-CoV-2 genome sequence identified as the B.1.1.7 variant of concern from a Canadian municipality concurrent with an outbreak

An Amplicon-Based Approach for the Whole-Genome Sequencing of Human Metapneumovirus

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