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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2021-01-01
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| author | Joseph Ojonugwa Shaibu Chika K Onwuamah Ayorinde Babatunde James Azuka Patrick Okwuraiwe Olufemi Samuel Amoo Olumuyiwa B Salu Fehintola A Ige Gideon Liboro Ebenezer Odewale Leona Chika Okoli Rahaman A Ahmed Dominic Achanya Adesegun Adesesan Oyewunmi Abosede Amuda Judith Sokei Bola A O Oyefolu Babatunde Lawal Salako Sunday Aremu Omilabu Rosemary Ajuma Audu |
| author_facet | Joseph Ojonugwa Shaibu Chika K Onwuamah Ayorinde Babatunde James Azuka Patrick Okwuraiwe Olufemi Samuel Amoo Olumuyiwa B Salu Fehintola A Ige Gideon Liboro Ebenezer Odewale Leona Chika Okoli Rahaman A Ahmed Dominic Achanya Adesegun Adesesan Oyewunmi Abosede Amuda Judith Sokei Bola A O Oyefolu Babatunde Lawal Salako Sunday Aremu Omilabu Rosemary Ajuma Audu |
| author_sort | Joseph Ojonugwa Shaibu |
| collection | DOAJ |
| 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. |
| format | Article |
| id | doaj-art-30ed2a12232f491da8851568e0dc0314 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2021-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-30ed2a12232f491da8851568e0dc03142025-08-20T02:55:13ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01161e024327110.1371/journal.pone.0243271Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria.Joseph Ojonugwa ShaibuChika K OnwuamahAyorinde Babatunde JamesAzuka Patrick OkwuraiweOlufemi Samuel AmooOlumuyiwa B SaluFehintola A IgeGideon LiboroEbenezer OdewaleLeona Chika OkoliRahaman A AhmedDominic AchanyaAdesegun AdesesanOyewunmi Abosede AmudaJudith SokeiBola A O OyefoluBabatunde Lawal SalakoSunday Aremu OmilabuRosemary Ajuma AuduIn 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.https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0243271&type=printable |
| spellingShingle | Joseph Ojonugwa Shaibu Chika K Onwuamah Ayorinde Babatunde James Azuka Patrick Okwuraiwe Olufemi Samuel Amoo Olumuyiwa B Salu Fehintola A Ige Gideon Liboro Ebenezer Odewale Leona Chika Okoli Rahaman A Ahmed Dominic Achanya Adesegun Adesesan Oyewunmi Abosede Amuda Judith Sokei Bola A O Oyefolu Babatunde Lawal Salako Sunday Aremu Omilabu Rosemary Ajuma Audu Full length genomic sanger sequencing and phylogenetic analysis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Nigeria. PLoS ONE |
| 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 |
| url | https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0243271&type=printable |
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