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Simple protocol for population (Sanger) sequencing for Zika virus genomic regions
BACKGROUND: A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTI...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Instituto Oswaldo Cruz, Ministério da Saúde
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719533/ https://www.ncbi.nlm.nih.gov/pubmed/29185594 http://dx.doi.org/10.1590/0074-02760170248 |
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author | Cabral, Gabriela Bastos Ferreira, João Leandro de Paula de Souza, Renato Pereira Cunha, Mariana Sequetin Luchs, Adriana Figueiredo, Cristina Adelaide Brígido, Luís Fernando de Macedo |
author_facet | Cabral, Gabriela Bastos Ferreira, João Leandro de Paula de Souza, Renato Pereira Cunha, Mariana Sequetin Luchs, Adriana Figueiredo, Cristina Adelaide Brígido, Luís Fernando de Macedo |
author_sort | Cabral, Gabriela Bastos |
collection | PubMed |
description | BACKGROUND: A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTIVE: The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. METHODS: Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. FINDINGS: Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. MAIN CONCLUSIONS: The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution. |
format | Online Article Text |
id | pubmed-5719533 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | Instituto Oswaldo Cruz, Ministério da Saúde |
record_format | MEDLINE/PubMed |
spelling | pubmed-57195332018-01-01 Simple protocol for population (Sanger) sequencing for Zika virus genomic regions Cabral, Gabriela Bastos Ferreira, João Leandro de Paula de Souza, Renato Pereira Cunha, Mariana Sequetin Luchs, Adriana Figueiredo, Cristina Adelaide Brígido, Luís Fernando de Macedo Mem Inst Oswaldo Cruz Articles BACKGROUND: A number of Zika virus (ZIKV) sequences were obtained using Next-generation sequencing (NGS), a methodology widely applied in genetic diversity studies and virome discovery. However Sanger method is still a robust, affordable, rapid and specific tool to obtain valuable sequences. OBJECTIVE: The aim of this study was to develop a simple and robust Sanger sequencing protocol targeting ZIKV relevant genetic regions, as envelope protein and nonstructural protein 5 (NS5). In addition, phylogenetic analysis of the ZIKV strains obtained using the present protocol and their comparison with previously published NGS sequences were also carried out. METHODS: Six Vero cells isolates from serum and one urine sample were available to develop the procedure. Primer sets were designed in order to conduct a nested RT-PCR and a Sanger sequencing protocols. Bayesian analysis was used to infer phylogenetic relationships. FINDINGS: Seven complete ZIKV envelope protein (1,571 kb) and six partial NS5 (0,798 Kb) were obtained using the protocol, with no amplification of NS5 gene from urine sample. Two NS5 sequences presented ambiguities at positions 495 and 196. Nucleotide analysis of a Sanger sequence and consensus sequence of previously NGS study revealed 100% identity. ZIKV strains described here clustered within the Asian lineage. MAIN CONCLUSIONS: The present study provided a simple and low-cost Sanger protocol to sequence relevant genes of the ZIKV genome. The identity of Sanger generated sequences with published consensus NGS support the use of Sanger method for ZIKV population studies. The regions evaluated were able to provide robust phylogenetic signals and may be used to conduct molecular epidemiological studies and monitor viral evolution. Instituto Oswaldo Cruz, Ministério da Saúde 2017-11-27 2018-01 /pmc/articles/PMC5719533/ /pubmed/29185594 http://dx.doi.org/10.1590/0074-02760170248 Text en http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Articles Cabral, Gabriela Bastos Ferreira, João Leandro de Paula de Souza, Renato Pereira Cunha, Mariana Sequetin Luchs, Adriana Figueiredo, Cristina Adelaide Brígido, Luís Fernando de Macedo Simple protocol for population (Sanger) sequencing for Zika virus genomic regions |
title | Simple protocol for population (Sanger) sequencing for Zika virus
genomic regions |
title_full | Simple protocol for population (Sanger) sequencing for Zika virus
genomic regions |
title_fullStr | Simple protocol for population (Sanger) sequencing for Zika virus
genomic regions |
title_full_unstemmed | Simple protocol for population (Sanger) sequencing for Zika virus
genomic regions |
title_short | Simple protocol for population (Sanger) sequencing for Zika virus
genomic regions |
title_sort | simple protocol for population (sanger) sequencing for zika virus
genomic regions |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719533/ https://www.ncbi.nlm.nih.gov/pubmed/29185594 http://dx.doi.org/10.1590/0074-02760170248 |
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