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. OBJECT...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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Fundação Oswaldo Cruz (FIOCRUZ)
2017-11-01
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| Series: | Memorias do Instituto Oswaldo Cruz |
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| Online Access: | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000100038&lng=en&tlng=en |
| _version_ | 1797721296954458112 |
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| author | Gabriela Bastos Cabral João Leandro de Paula Ferreira Renato Pereira de Souza Mariana Sequetin Cunha Adriana Luchs Cristina Adelaide Figueiredo Luís Fernando de Macedo Brígido |
| author_facet | Gabriela Bastos Cabral João Leandro de Paula Ferreira Renato Pereira de Souza Mariana Sequetin Cunha Adriana Luchs Cristina Adelaide Figueiredo Luís Fernando de Macedo Brígido |
| author_sort | Gabriela Bastos Cabral |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-03-12T09:31:25Z |
| format | Article |
| id | doaj.art-b18d01008f244421ba6b9f3c2fb2704a |
| institution | Directory Open Access Journal |
| issn | 1678-8060 |
| language | English |
| last_indexed | 2024-03-12T09:31:25Z |
| publishDate | 2017-11-01 |
| publisher | Fundação Oswaldo Cruz (FIOCRUZ) |
| record_format | Article |
| series | Memorias do Instituto Oswaldo Cruz |
| spelling | doaj.art-b18d01008f244421ba6b9f3c2fb2704a2023-09-02T13:54:10ZengFundação Oswaldo Cruz (FIOCRUZ)Memorias do Instituto Oswaldo Cruz1678-80602017-11-011131384410.1590/0074-02760170248S0074-02762018000100038Simple protocol for population (Sanger) sequencing for Zika virus genomic regionsGabriela Bastos CabralJoão Leandro de Paula FerreiraRenato Pereira de SouzaMariana Sequetin CunhaAdriana LuchsCristina Adelaide FigueiredoLuís Fernando de Macedo BrígidoBACKGROUND 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.http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000100038&lng=en&tlng=enZika virusSanger sequencingPCRphylogeny |
| spellingShingle | Gabriela Bastos Cabral João Leandro de Paula Ferreira Renato Pereira de Souza Mariana Sequetin Cunha Adriana Luchs Cristina Adelaide Figueiredo Luís Fernando de Macedo Brígido Simple protocol for population (Sanger) sequencing for Zika virus genomic regions Memorias do Instituto Oswaldo Cruz Zika virus Sanger sequencing PCR phylogeny |
| 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 | Zika virus Sanger sequencing PCR phylogeny |
| url | http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762018000100038&lng=en&tlng=en |
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