Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria

Abstract

In an outbreak, effective detection of the aetiological agent(s) involved using molecular techniques is key to efficient diagnosis, early prevention and management of the spread. However, sequencing is necessary for mutation monitoring and tracking of clusters of transmission, development of diagnostics and for vaccines and drug development. Many sequencing methods are fast evolving to reduce test turn-around-time and to increase through-put compared to Sanger sequencing method; however, Sanger sequencing remains the gold standard for clinical research sequencing with its 99.99% accuracy This study sought to generate sequence data of SARS-CoV-2 using Sanger sequencing method and to characterize them for possible site(s) of mutations. About 30 pairs of primers were designed, synthesized, and optimized using endpoint PCR to generate amplicons for the full length of the virus. Cycle sequencing using BigDye Terminator v.3.1 and capillary gel electrophoresis on ABI 3130xl genetic analyser were performed according to the manufacturers’ instructions. The sequence data generated were assembled and analysed for variations using DNASTAR Lasergene 17 SeqMan Ultra. Total length of 29,760bp of SARS-CoV-2 was assembled from the sample analysed and deposited in GenBank with accession number: MT576584. Blast result of the sequence assembly shows a 99.97% identity with the reference sequence. Variations were noticed at positions: nt201, nt2997, nt14368, nt16535, nt20334, and nt28841-28843, which caused amino acid alterations at the S (aa614) and N (aa203-204) regions. The mutations observed at S and N-gene in this study may be indicative of a gradual changes in the genetic coding of the virus hence, the need for active surveillance of the viral genome.

Molecular dynamics simulation study reveals effects of key mutations on spike protein structure in SARS-CoV-2

“…In the case of SARS-CoV 7 , the P462L mutation of S protein can achieve the escape of human mAb Cr3014 8 . Recently, the United Kingdom, South Africa, Nigeria, and other places have reported the emergence and prevalence of mutant strains [9][10][11][12] . More disturbingly, a SARS-CoV-2 variant that achieved in vitro escapement from highly neutralizing COVID-19 convalescent plasma has been reported 13 .…”

SARS-CoV-2 variant detection with ADSSpike

“…SARS-CoV-2 VOC detection using WGS by capillary sequencing or NGS has been widely used for surveillance ( Goncalves Cabecinhas et al, 2021 , Pillay et al, 2020 , Miller et al, 2020 , Shaibu et al, 2021 ). In contrast to WGS, amplicon deep sequencing focuses on a specific gene or genomic region, providing a greater sequencing depth and coverage while reducing costs and data burden.…”

SARS‐CoV‐2 Delta variant in Cartagena de Indias, Colombia, August 2021

“…Sanger sequencing of the spike gene is a rapid and low-cost strategy that allows it to identify signature mutations, and to distinguish and monitor circulating variants. 10,11 In this study, the mutation calling and Nextstrain clade assignment were successfully performed using [13][14][15] The cases described herein illustrate cryptic transmission of the Delta variant in Cartagena residents with no travel history. However, since subjects were recruited through passive surveillance, we are unable to determine community transmission.…”