Amplicon-based detection and sequencing of SARS-CoV-2 in nasopharyngeal swabs from patients with COVID-19 and identification of deletions in the viral genome that encode proteins involved in interferon antagonism
Author(s)
Type
Journal Article
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Sequencing the viral genome as the outbreak progresses is important, particularly in the identification of emerging isolates with different pathogenic potential and to identify whether nucleotide changes in the genome will impair clinical diagnostic tools such as real-time PCR assays. Although single nucleotide polymorphisms and point mutations occur during the replication of coronaviruses, one of the biggest drivers in genetic change is recombination. This can manifest itself in insertions and/or deletions in the viral genome. Therefore, sequencing strategies that underpin molecular epidemiology and inform virus biology in patients should take these factors into account. A long amplicon/read length-based RT-PCR sequencing approach focused on the Oxford Nanopore MinION/GridION platforms was developed to identify and sequence the SARS-CoV-2 genome in samples from patients with or suspected of COVID-19. The protocol, termed Rapid Sequencing Long Amplicons (RSLAs) used random primers to generate cDNA from RNA purified from a sample from a patient, followed by single or multiplex PCRs to generate longer amplicons of the viral genome. The base protocol was used to identify SARS-CoV-2 in a variety of clinical samples and proved sensitive in identifying viral RNA in samples from patients that had been declared negative using other nucleic acid-based assays (false negative). Sequencing the amplicons revealed that a number of patients had a proportion of viral genomes with deletions.
Date Issued
2020-10-14
Date Acceptance
2020-10-09
Citation
Viruses, 2020, 12, pp.1-16
ISSN
1999-4915
Start Page
1
End Page
16
Journal / Book Title
Viruses
Volume
12
Copyright Statement
© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
License URL
Sponsor
Wellcome Trust
Wellcome Trust
Imperial College Healthcare NHS Trust- BRC Funding
National Institute for Health Research
National Institute for Health Research
Medical Research Council (MRC)
National Institute for Health Research
Wellcome Trust
National Institute for Health Research
UKRI MRC COVID-19 Rapid Response Call
Identifier
https://www.mdpi.com/1999-4915/12/10/1164
Grant Number
090382/Z/09/Z
090382/Z/09/Z
RDA06 79560
NF-SI-0513-10150
HPRU-2012-10064
MR/R502121/1
RDF04
214407/Z/18/Z
NIHR201385
MC_PC19025
Subjects
MinION
SARS-CoV-2
amplicon
next-generation sequencing
Betacoronavirus
Clinical Laboratory Techniques
Coronavirus Infections
DNA, Complementary
DNA, Viral
Genome, Viral
High-Throughput Nucleotide Sequencing
Humans
Molecular Epidemiology
Multiplex Polymerase Chain Reaction
Pandemics
Pneumonia, Viral
RNA, Viral
Real-Time Polymerase Chain Reaction
Sequence Analysis
Humans
Pneumonia, Viral
Coronavirus Infections
DNA, Complementary
DNA, Viral
RNA, Viral
Clinical Laboratory Techniques
Sequence Analysis
Genome, Viral
Molecular Epidemiology
Pandemics
High-Throughput Nucleotide Sequencing
Real-Time Polymerase Chain Reaction
Multiplex Polymerase Chain Reaction
Betacoronavirus
0605 Microbiology
Notes
article-number: 1164
Publication Status
Published
Article Number
10
Date Publish Online
2020-10-14