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© 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/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

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.

Details

Title
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
Moore, Shona C 1 ; Penrice-Randal, Rebekah 1   VIAFID ORCID Logo  ; Alruwaili, Muhannad 1   VIAFID ORCID Logo  ; Randle, Nadine 1   VIAFID ORCID Logo  ; Armstrong, Stuart 1 ; Hartley, Catherine 1   VIAFID ORCID Logo  ; Haldenby, Sam 1 ; Dong, Xiaofeng 1 ; Alrezaihi, Abdulrahman 1   VIAFID ORCID Logo  ; Almsaud, Mai 1 ; Bentley, Eleanor 1 ; Clark, Jordan 1   VIAFID ORCID Logo  ; García-Dorival, Isabel 1   VIAFID ORCID Logo  ; Gilmore, Paul 1 ; Han, Ximeng 1 ; Jones, Benjamin 1 ; Luu, Lisa 1   VIAFID ORCID Logo  ; Sharma, Parul 1   VIAFID ORCID Logo  ; Shawli, Ghada 1 ; Sun, Yani 2 ; Zhao, Qin 2 ; Pullan, Steven T 3 ; Carter, Daniel P 3 ; Bewley, Kevin 3 ; Dunning, Jake 4   VIAFID ORCID Logo  ; En-min, Zhou 5 ; Solomon, Tom 6 ; Beadsworth, Michael 7 ; Cruise, James 7 ; Crook, Derrick W 8 ; Matthews, David A 9 ; Davidson, Andrew D 9 ; Mahmood, Zana 10 ; Aljabr, Waleed 11   VIAFID ORCID Logo  ; Druce, Julian 12 ; Vipond, Richard 4 ; Ng, Lisa 13   VIAFID ORCID Logo  ; Laurent Renia 14   VIAFID ORCID Logo  ; Openshaw, Peter J M 15 ; Baillie, J Kenneth 16 ; Carroll, Miles W 17 ; Stewart, James 18   VIAFID ORCID Logo  ; Darby, Alistair 18 ; Semple, Malcolm 6   VIAFID ORCID Logo  ; Turtle, Lance 6 ; Hiscox, Julian A 13 

 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK; [email protected] (S.C.M.); [email protected] (R.P.-R.); [email protected] (M.A); [email protected] (N.R.); [email protected] (S.A.); [email protected] (C.H.); [email protected] (S.H.); [email protected] (X.D.); [email protected] (A.A.); [email protected] (M.A.); [email protected] (E.B.); [email protected] (J.C.); [email protected] (I.G.-D.); [email protected] (P.G.); [email protected] (X.H.); [email protected] (B.J.); [email protected] (L.L.); [email protected] (P.S.); [email protected] (G.S.); [email protected] (Y.S.); [email protected] (Q.Z.); [email protected] (T.S.); [email protected] (Z.M.); [email protected] (W.A.); [email protected] (L.N.); [email protected] (J.S.); [email protected] (A.D.); [email protected] (M.S.) 
 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK; [email protected] (S.C.M.); [email protected] (R.P.-R.); [email protected] (M.A); [email protected] (N.R.); [email protected] (S.A.); [email protected] (C.H.); [email protected] (S.H.); [email protected] (X.D.); [email protected] (A.A.); [email protected] (M.A.); [email protected] (E.B.); [email protected] (J.C.); [email protected] (I.G.-D.); [email protected] (P.G.); [email protected] (X.H.); [email protected] (B.J.); [email protected] (L.L.); [email protected] (P.S.); [email protected] (G.S.); [email protected] (Y.S.); [email protected] (Q.Z.); [email protected] (T.S.); [email protected] (Z.M.); [email protected] (W.A.); [email protected] (L.N.); [email protected] (J.S.); [email protected] (A.D.); [email protected] (M.S.); College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China; [email protected] 
 National Infection Service, Public Health England, Salisbury SP4 0JG, UK; [email protected] (S.T.P.); [email protected] (D.P.C.); [email protected] (K.B.); [email protected] (J.D.); [email protected] (R.V.); [email protected] (M.W.C.) 
 National Infection Service, Public Health England, Salisbury SP4 0JG, UK; [email protected] (S.T.P.); [email protected] (D.P.C.); [email protected] (K.B.); [email protected] (J.D.); [email protected] (R.V.); [email protected] (M.W.C.); Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, UK 
 College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China; [email protected] 
 Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK; [email protected] (S.C.M.); [email protected] (R.P.-R.); [email protected] (M.A); [email protected] (N.R.); [email protected] (S.A.); [email protected] (C.H.); [email protected] (S.H.); [email protected] (X.D.); [email protected] (A.A.); [email protected] (M.A.); [email protected] (E.B.); [email protected] (J.C.); [email protected] (I.G.-D.); [email protected] (P.G.); [email protected] (X.H.); [email protected] (B.J.); [email protected] (L.L.); [email protected] (P.S.); [email protected] (G.S.); [email protected] (Y.S.); [email protected] (Q.Z.); [email protected] (T.S.); [email protected] (Z.M.); [email protected] (W.A.); [email protected] (L.N.); [email protected] (J.S.); [email protected] (A.D.); [email protected] (M.S.); Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, UK; Liverpool Health Partners, Liverpool L3 5TF, UK 
 Tropical & Infectious Disease Unit, Liverpool University Hospitals NHS Foundation Trust (a member of Liverpool Health Partners), Liverpool L7 8XP, UK; [email protected] (M.B.); [email protected] (J.C.) 
 Nuffield Department of Medicine, University of Oxford, Oxford OX3 7BN, UK; [email protected] 
 School of Cellular and Molecular Medicine, University of Bristol, Bristol BS8 1TD, UK; [email protected] (D.A.M.); [email protected] (A.D.D.) 
10  Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK; [email protected] (S.C.M.); [email protected] (R.P.-R.); [email protected] (M.A); [email protected] (N.R.); [email protected] (S.A.); [email protected] (C.H.); [email protected] (S.H.); [email protected] (X.D.); [email protected] (A.A.); [email protected] (M.A.); [email protected] (E.B.); [email protected] (J.C.); [email protected] (I.G.-D.); [email protected] (P.G.); [email protected] (X.H.); [email protected] (B.J.); [email protected] (L.L.); [email protected] (P.S.); [email protected] (G.S.); [email protected] (Y.S.); [email protected] (Q.Z.); [email protected] (T.S.); [email protected] (Z.M.); [email protected] (W.A.); [email protected] (L.N.); [email protected] (J.S.); [email protected] (A.D.); [email protected] (M.S.); Laboratory Department, Directorate of Veterinary in Sulaimany, Rizgari 402, Sulaimani, Kurdistan Region 46001, Iraq 
11  Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK; [email protected] (S.C.M.); [email protected] (R.P.-R.); [email protected] (M.A); [email protected] (N.R.); [email protected] (S.A.); [email protected] (C.H.); [email protected] (S.H.); [email protected] (X.D.); [email protected] (A.A.); [email protected] (M.A.); [email protected] (E.B.); [email protected] (J.C.); [email protected] (I.G.-D.); [email protected] (P.G.); [email protected] (X.H.); [email protected] (B.J.); [email protected] (L.L.); [email protected] (P.S.); [email protected] (G.S.); [email protected] (Y.S.); [email protected] (Q.Z.); [email protected] (T.S.); [email protected] (Z.M.); [email protected] (W.A.); [email protected] (L.N.); [email protected] (J.S.); [email protected] (A.D.); [email protected] (M.S.); Research Center, King Fahad Medical City, Riyadh 11525, Saudi Arabia 
12  The Peter Doherty Institute for Infection and Immunity, Melbourne 3000, Australia; [email protected] 
13  Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK; [email protected] (S.C.M.); [email protected] (R.P.-R.); [email protected] (M.A); [email protected] (N.R.); [email protected] (S.A.); [email protected] (C.H.); [email protected] (S.H.); [email protected] (X.D.); [email protected] (A.A.); [email protected] (M.A.); [email protected] (E.B.); [email protected] (J.C.); [email protected] (I.G.-D.); [email protected] (P.G.); [email protected] (X.H.); [email protected] (B.J.); [email protected] (L.L.); [email protected] (P.S.); [email protected] (G.S.); [email protected] (Y.S.); [email protected] (Q.Z.); [email protected] (T.S.); [email protected] (Z.M.); [email protected] (W.A.); [email protected] (L.N.); [email protected] (J.S.); [email protected] (A.D.); [email protected] (M.S.); Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, UK; Liverpool Health Partners, Liverpool L3 5TF, UK; Infectious Diseases Horizontal Technology Centre (ID HTC), A*STAR, Singapore 138648, Singapore; [email protected] 
14  Infectious Diseases Horizontal Technology Centre (ID HTC), A*STAR, Singapore 138648, Singapore; [email protected] 
15  Faculty of Medicine, Imperial College London, London SW7 2AZ, UK; [email protected] 
16  Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, UK; [email protected] 
17  National Infection Service, Public Health England, Salisbury SP4 0JG, UK; [email protected] (S.T.P.); [email protected] (D.P.C.); [email protected] (K.B.); [email protected] (J.D.); [email protected] (R.V.); [email protected] (M.W.C.); Health Protection Research Unit in Emerging and Zoonotic Infections, Liverpool L69 7BE, UK; Nuffield Department of Medicine, University of Oxford, Oxford OX3 7BN, UK; [email protected] 
18  Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L3 5RF, UK; [email protected] (S.C.M.); [email protected] (R.P.-R.); [email protected] (M.A); [email protected] (N.R.); [email protected] (S.A.); [email protected] (C.H.); [email protected] (S.H.); [email protected] (X.D.); [email protected] (A.A.); [email protected] (M.A.); [email protected] (E.B.); [email protected] (J.C.); [email protected] (I.G.-D.); [email protected] (P.G.); [email protected] (X.H.); [email protected] (B.J.); [email protected] (L.L.); [email protected] (P.S.); [email protected] (G.S.); [email protected] (Y.S.); [email protected] (Q.Z.); [email protected] (T.S.); [email protected] (Z.M.); [email protected] (W.A.); [email protected] (L.N.); [email protected] (J.S.); [email protected] (A.D.); [email protected] (M.S.); Liverpool Health Partners, Liverpool L3 5TF, UK 
First page
1164
Publication year
2020
Publication date
2020
Publisher
MDPI AG
e-ISSN
19994915
Source type
Scholarly Journal
Language of publication
English
ProQuest document ID
2550295591
Copyright
© 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/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.