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
| Autor: | Sam Haldenby, Michael Beadsworth, Julian Druce, Catherine Hartley, David A. Matthews, Benjamin Jones, Lisa Luu, Isabel García-Dorival, Ximeng Han, Malcolm G Semple, Peter J. M. Openshaw, Jake Dunning, Shona C Moore, Miles W. Carroll, Qin Zhao, Paul Gilmore, Nadine Randle, Xiaofeng Dong, Kevin R. Bewley, Laurent Renia, Rebekah Penrice-Randal, James Cruise, Lisa Ng, Waleed Aljabr, Steven T. Pullan, James P. Stewart, J Kenneth Baillie, Parul Sharma, Alistair C. Darby, Tom Solomon, Stuart D. Armstrong, Muhannad Alruwaili, Eleanor G. Bentley, Mai Almsaud, Derrick W. Crook, Julian A. Hiscox, Lance Turtle, Abdulrahman Alrezaihi, Yani Sun, Andrew D. Davidson, En-Min Zhou, Ghada Shawli, Daniel P. Carter, Jordan J. Clark, Zana H. Mahmood, Richard Vipond |
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| Přispěvatelé: | Wellcome Trust, Imperial College Healthcare NHS Trust- BRC Funding, National Institute for Health Research, Medical Research Council (MRC), UKRI MRC COVID-19 Rapid Response Call |
| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
COVID-19 Vaccines DNA Complementary Sequence analysis viruses Pneumonia Viral lcsh:QR1-502 MinION Single-nucleotide polymorphism Genome Viral Real-Time Polymerase Chain Reaction Genome Article lcsh:Microbiology DNA sequencing Betacoronavirus 03 medical and health sciences COVID-19 Testing 0302 clinical medicine Virology Multiplex polymerase chain reaction Humans SARS-CoV-2 next-generation sequencing amplicon 030212 general & internal medicine Pandemics Genetics Molecular Epidemiology biology Clinical Laboratory Techniques COVID-19 High-Throughput Nucleotide Sequencing Amplicon biology.organism_classification 030104 developmental biology Infectious Diseases DNA Viral RNA Viral Nanopore sequencing Coronavirus Infections Multiplex Polymerase Chain Reaction Sequence Analysis 0605 Microbiology |
| Zdroj: | Viruses, Vol 12, Iss 1164, p 1164 (2020) Viruses; Volume 12; Issue 10; Pages: 1164 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 2020, ' 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, vol. 12, no. 10 . https://doi.org/10.3390/v12101164 VIRUSES-BASEL Viruses |
| ISSN: | 1999-4915 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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