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

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 diagnos...

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Autores principales: Shaibu, Joseph Ojonugwa, Onwuamah, Chika K., James, Ayorinde Babatunde, Okwuraiwe, Azuka Patrick, Amoo, Olufemi Samuel, Salu, Olumuyiwa B., Ige, Fehintola A., Liboro, Gideon, Odewale, Ebenezer, Okoli, Leona Chika, Ahmed, Rahaman A., Achanya, Dominic, Adesesan, Adesegun, Amuda, Oyewunmi Abosede, Sokei, Judith, Oyefolu, Bola A. O., Salako, Babatunde Lawal, Omilabu, Sunday Aremu, Audu, Rosemary Ajuma
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799769/
https://www.ncbi.nlm.nih.gov/pubmed/33428634
http://dx.doi.org/10.1371/journal.pone.0243271
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author Shaibu, Joseph Ojonugwa
Onwuamah, Chika K.
James, Ayorinde Babatunde
Okwuraiwe, Azuka Patrick
Amoo, Olufemi Samuel
Salu, Olumuyiwa B.
Ige, Fehintola A.
Liboro, Gideon
Odewale, Ebenezer
Okoli, Leona Chika
Ahmed, Rahaman A.
Achanya, Dominic
Adesesan, Adesegun
Amuda, Oyewunmi Abosede
Sokei, Judith
Oyefolu, Bola A. O.
Salako, Babatunde Lawal
Omilabu, Sunday Aremu
Audu, Rosemary Ajuma
author_facet Shaibu, Joseph Ojonugwa
Onwuamah, Chika K.
James, Ayorinde Babatunde
Okwuraiwe, Azuka Patrick
Amoo, Olufemi Samuel
Salu, Olumuyiwa B.
Ige, Fehintola A.
Liboro, Gideon
Odewale, Ebenezer
Okoli, Leona Chika
Ahmed, Rahaman A.
Achanya, Dominic
Adesesan, Adesegun
Amuda, Oyewunmi Abosede
Sokei, Judith
Oyefolu, Bola A. O.
Salako, Babatunde Lawal
Omilabu, Sunday Aremu
Audu, Rosemary Ajuma
author_sort Shaibu, Joseph Ojonugwa
collection PubMed
description 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.
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spelling pubmed-77997692021-01-22 Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria Shaibu, Joseph Ojonugwa Onwuamah, Chika K. James, Ayorinde Babatunde Okwuraiwe, Azuka Patrick Amoo, Olufemi Samuel Salu, Olumuyiwa B. Ige, Fehintola A. Liboro, Gideon Odewale, Ebenezer Okoli, Leona Chika Ahmed, Rahaman A. Achanya, Dominic Adesesan, Adesegun Amuda, Oyewunmi Abosede Sokei, Judith Oyefolu, Bola A. O. Salako, Babatunde Lawal Omilabu, Sunday Aremu Audu, Rosemary Ajuma PLoS One Research Article 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. Public Library of Science 2021-01-11 /pmc/articles/PMC7799769/ /pubmed/33428634 http://dx.doi.org/10.1371/journal.pone.0243271 Text en © 2021 Shaibu et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Shaibu, Joseph Ojonugwa
Onwuamah, Chika K.
James, Ayorinde Babatunde
Okwuraiwe, Azuka Patrick
Amoo, Olufemi Samuel
Salu, Olumuyiwa B.
Ige, Fehintola A.
Liboro, Gideon
Odewale, Ebenezer
Okoli, Leona Chika
Ahmed, Rahaman A.
Achanya, Dominic
Adesesan, Adesegun
Amuda, Oyewunmi Abosede
Sokei, Judith
Oyefolu, Bola A. O.
Salako, Babatunde Lawal
Omilabu, Sunday Aremu
Audu, Rosemary Ajuma
Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
title Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
title_full Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
title_fullStr Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
title_full_unstemmed Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
title_short Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria
title_sort full length genomic sanger sequencing and phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) in nigeria
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7799769/
https://www.ncbi.nlm.nih.gov/pubmed/33428634
http://dx.doi.org/10.1371/journal.pone.0243271
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