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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Funding: This work was supported by the United States Food and Drug Administration grant number HHSF223201510104C ‘Ebola Virus Disease: correlates of protection, determinants of outcome and clinical management’ amended to incorporate urgent COVID-19 studies. Awarded to J.A.H., A.D., D.A.M. and M.W.C. Recruitment, sample acquisition, transport, laboratory and management costs were supported by the Medical Research Council “Protocol for Severe Emerging Infection” grant number MC_PC_19025 awarded to J.K.B., M.G.S., and P.J.M.O. This work was supported by Research Center, King Fahad Medical City, Saudi Arabia grant number 019-003 ‘Elucidating the viral biology of MERS-CoV and the host response using high resolution sequencing’. Awarded to W.A. This work was supported by the Medical Research Council Discovery Medicine North Doctoral Training Partnership and directly funded the studentship of R.P.-R.This work was supported by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections at University of Liverpool in partnership with Public Health England (PHE), in collaboration with Liverpool School of Tropical Medicine. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England. Awarded to T.S. This work is also supported by the Centre of Excellence in Infectious Diseases Research (CEIDR) and the Alder Hey Charity awarded to J.A.H. and L.T. We also acknowledge support of Liverpool Health Partners and the Liverpool-Malawi-Covid-19 Consortium. L.T. is a Wellcome Trust clinical career development fellow, supported by grant number 205228/Z/16/Z.
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- Next-generation sequencing