tetano
Editor, Senior Moderator
PLoS One
. 2021 Jan 11;16(1):e0243271.
doi: 10.1371/journal.pone.0243271. eCollection 2021.
Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
Joseph Ojonugwa Shaibu[SUP] 1 [/SUP], Chika K Onwuamah[SUP] 1 [/SUP], Ayorinde Babatunde James[SUP] 2 [/SUP], Azuka Patrick Okwuraiwe[SUP] 1 [/SUP], Olufemi Samuel Amoo[SUP] 1 [/SUP], Olumuyiwa B Salu[SUP] 3 [/SUP], Fehintola A Ige[SUP] 1 [/SUP], Gideon Liboro[SUP] 1 [/SUP], Ebenezer Odewale[SUP] 1 [/SUP], Leona Chika Okoli[SUP] 1 [/SUP], Rahaman A Ahmed[SUP] 1 4 [/SUP], Dominic Achanya[SUP] 1 [/SUP], Adesegun Adesesan[SUP] 5 [/SUP], Oyewunmi Abosede Amuda[SUP] 5 [/SUP], Judith Sokei[SUP] 1 [/SUP], Bola A O Oyefolu[SUP] 6 [/SUP], Babatunde Lawal Salako[SUP] 7 [/SUP], Sunday Aremu Omilabu[SUP] 1 3 [/SUP], Rosemary Ajuma Audu[SUP] 1 [/SUP]
Affiliations
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.
. 2021 Jan 11;16(1):e0243271.
doi: 10.1371/journal.pone.0243271. eCollection 2021.
Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
Joseph Ojonugwa Shaibu[SUP] 1 [/SUP], Chika K Onwuamah[SUP] 1 [/SUP], Ayorinde Babatunde James[SUP] 2 [/SUP], Azuka Patrick Okwuraiwe[SUP] 1 [/SUP], Olufemi Samuel Amoo[SUP] 1 [/SUP], Olumuyiwa B Salu[SUP] 3 [/SUP], Fehintola A Ige[SUP] 1 [/SUP], Gideon Liboro[SUP] 1 [/SUP], Ebenezer Odewale[SUP] 1 [/SUP], Leona Chika Okoli[SUP] 1 [/SUP], Rahaman A Ahmed[SUP] 1 4 [/SUP], Dominic Achanya[SUP] 1 [/SUP], Adesegun Adesesan[SUP] 5 [/SUP], Oyewunmi Abosede Amuda[SUP] 5 [/SUP], Judith Sokei[SUP] 1 [/SUP], Bola A O Oyefolu[SUP] 6 [/SUP], Babatunde Lawal Salako[SUP] 7 [/SUP], Sunday Aremu Omilabu[SUP] 1 3 [/SUP], Rosemary Ajuma Audu[SUP] 1 [/SUP]
Affiliations
- PMID: 33428634
- DOI: 10.1371/journal.pone.0243271
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.