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

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.

Current and innovative methods for the diagnosis of COVID‑19 infection (Review)

“…In particular, metagenomic sequencing allows the identification of the normal microbiome of patients, while amplicon-based sequencing allows the amplification and the subsequent sequencing of SARS-CoV-2 viral RNA. Together, amplicon-based sequencing and metagenomic sequencing are able to correctly diagnose COVID-19 infection, thus also identifying secondary infections due to other pathogens aggravating the health status of patients ( 109 ).…”

Covid-19: variants and vaccination

“…SARS-CoV-2 infections can be established by multiple virus genotypes within the same person,7 and together with new rounds of virus replication, provide the raw material for natural selection. The extent of genetic diversity in a viral population is critical to natural selection for growth advantages such as better binding to the receptor, faster replication, and more effective suppression or avoidance of the host immune response.…”

Sequencing of SARS-CoV-2 genome using different nanopore chemistries

“…Despite of the larger number of mismatches from R10, the consensus sequence resulted in the same variants detected as from R9. Previous attempts to sequence the SARS-CoV-2 genome have provided accurate results for new variants discovery using rapid workflows based on the ARTIC protocol (Chan et al 2020 ; Li et al 2020 ; Moore et al 2020 ). Other studies also showed amplicon sequencing using ONT flongle flow cells (Chan et al 2020 ).…”