期刊
VIRUSES-BASEL
卷 12, 期 10, 页码 -出版社
MDPI
DOI: 10.3390/v12101164
关键词
SARS-CoV-2; next-generation sequencing; amplicon; MinION
类别
资金
- United States Food and Drug Administration [HHSF223201510104C]
- Medical Research Council [MC_PC_19025]
- Research Center, King Fahad Medical City, Saudi Arabia [019-003]
- Medical Research Council Discovery Medicine North Doctoral Training Partnership
- National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Emerging and Zoonotic Infections at University of Liverpool
- Public Health England (PHE)
- Liverpool School of Tropical Medicine
- Centre of Excellence in Infectious Diseases Research (CEIDR)
- Alder Hey Charity
- Liverpool-Malawi-Covid-19 Consortium
- Liverpool Health Partners
- [205228/Z/16/Z]
- BBSRC [BB/M02542X/1] Funding Source: UKRI
- MRC [MC_PC_19025, MR/R502121/1, MR/V033441/1] Funding Source: UKRI
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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