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
by
Shona C. Moore 1,†, Rebekah Penrice-Randal 1,†
, Muhannad Alruwaili 1,†, Nadine Randle 1,†, Stuart Armstrong 1,†, Catherine Hartley 1,†, Sam Haldenby 1, Xiaofeng Dong 1, Abdulrahman Alrezaihi 1, Mai Almsaud 1, Eleanor Bentley 1, Jordan Clark 1, Isabel García-Dorival 1, Paul Gilmore 1, Ximeng Han 1, Benjamin Jones 1, Lisa Luu 1, Parul Sharma 1, Ghada Shawli 1, Yani Sun 1,2, Qin Zhao 1,2, Steven T. Pullan 3, Daniel P. Carter 3, Kevin Bewley 3, Jake Dunning 3,4, En-min Zhou 2, Tom Solomon 1,4,5, Michael Beadsworth 6, James Cruise 6, Derrick W. Crook 7, David A. Matthews 8, Andrew D. Davidson 8, Zana Mahmood 1,9, Waleed Aljabr 1,10, Julian Druce 11, Richard Vipond 3,4, Lisa Ng 1,4,5,12, Laurent Renia 12, Peter J. M. Openshaw 13, J. Kenneth Baillie 14, Miles W. Carroll 3,4,7, James Stewart 1,5, Alistair Darby 1,5, Malcolm Semple 1,4,5, Lance Turtle 1,4,5,* and Julian A. Hiscox 1,4,5,12,*
Shona C. Moore 1,†, Rebekah Penrice-Randal 1,†, Muhannad Alruwaili 1,†, Nadine Randle 1,†, Stuart Armstrong 1,†, Catherine Hartley 1,†, Sam Haldenby 1, Xiaofeng Dong 1, Abdulrahman Alrezaihi 1, Mai Almsaud 1, Eleanor Bentley 1, Jordan Clark 1, Isabel García-Dorival 1, Paul Gilmore 1, Ximeng Han 1, Benjamin Jones 1, Lisa Luu 1, Parul Sharma 1, Ghada Shawli 1, Yani Sun 1,2, Qin Zhao 1,2, Steven T. Pullan 3, Daniel P. Carter 3, Kevin Bewley 3, Jake Dunning 3,4, En-min Zhou 2, Tom Solomon 1,4,5, Michael Beadsworth 6, James Cruise 6, Derrick W. Crook 7, David A. Matthews 8, Andrew D. Davidson 8, Zana Mahmood 1,9, Waleed Aljabr 1,10, Julian Druce 11, Richard Vipond 3,4, Lisa Ng 1,4,5,12, Laurent Renia 12, Peter J. M. Openshaw 13, J. Kenneth Baillie 14, Miles W. Carroll 3,4,7, James Stewart 1,5, Alistair Darby 1,5, Malcolm Semple 1,4,5, Lance Turtle 1,4,5,* and Julian A. Hiscox 1,4,5,12,*
1
Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK
2
College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China
3
National Infection Service, Public Health England, Salisbury SP4 0JG, UK
4
Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, UK
5
Liverpool Health Partners, Liverpool L3 5TF, UK
6
Tropical & Infectious Disease Unit, Liverpool University Hospitals NHS Foundation Trust (a member of Liverpool Health Partners), Liverpool L7 8XP, UK
7
Nuffield Department of Medicine, University of Oxford, Oxford OX3 7BN, UK
8
School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK
9
Laboratory Department, Directorate of Veterinary in Sulaimany, Rizgari 402, Sulaimani, Kurdistan Region 46001, Iraq
10
Research Center, King Fahad Medical City, Riyadh 11525, Saudi Arabia
11
The Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia
12
Infectious Diseases Horizontal Technology Centre (ID HTC), A*STAR, Singapore 138648, Singapore
13
Faculty of Medicine, Imperial College London, London SW7 2AZ, UK
14
Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, UK
*
Authors to whom correspondence should be addressed.
†
These authors Contributed equally to this work.
Viruses 2020, 12(10), 1164; https://doi.org/10.3390/v12101164
Received: 8 September 2020 / Revised: 8 October 2020 / Accepted: 9 October 2020 / Published: 14 October 2020
(This article belongs to the Collection Coronaviruses)
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.
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Keywords:
SARS-CoV-2; next-generation sequencing; amplicon; MinION
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MDPI and ACS Style
Moore, S.C.; Penrice-Randal, R.; Alruwaili, M.; Randle, N.; Armstrong, S.; Hartley, C.; Haldenby, S.; Dong, X.; Alrezaihi, A.; Almsaud, M.; Bentley, E.; Clark, J.; García-Dorival, I.; Gilmore, P.; Han, X.; Jones, B.; Luu, L.; Sharma, P.; Shawli, G.; Sun, Y.; Zhao, Q.; Pullan, S.T.; Carter, D.P.; Bewley, K.; Dunning, J.; Zhou, E.-M.; Solomon, T.; Beadsworth, M.; Cruise, J.; Crook, D.W.; Matthews, D.A.; Davidson, A.D.; Mahmood, Z.; Aljabr, W.; Druce, J.; Vipond, R.; Ng, L.; Renia, L.; Openshaw, P.J.M.; Baillie, J.K.; Carroll, M.W.; Stewart, J.; Darby, A.; Semple, M.; Turtle, L.; Hiscox, J.A. 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. Viruses 2020, 12, 1164.
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