Simple protocol for population (Sanger) sequencing for Zika virus genomic regions
| Autor: | Adriana Luchs, Mariana Sequetin Cunha, Gabriela Bastos Cabral, Cristina Adelaide Figueiredo, João Leandro de Paula Ferreira, Renato Pereira de Souza, Luís Fernando de Macedo Brígido |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Microbiology (medical) Sanger sequencing lcsh:Arctic medicine. Tropical medicine lcsh:RC955-962 Population lcsh:QR1-502 Computational biology Genome Viral Viral Nonstructural Proteins phylogeny Genome lcsh:Microbiology Zika virus 03 medical and health sciences symbols.namesake Phylogenetics Consensus sequence Humans Human virome education Genetics Protocol (science) education.field_of_study Phylogenetic tree SIMPLE (military communications protocol) biology Reverse Transcriptase Polymerase Chain Reaction High-Throughput Nucleotide Sequencing Bayes Theorem General Medicine Articles Zika Virus biology.organism_classification 030104 developmental biology Infectious Diseases PCR symbols RNA Viral Primer (molecular biology) |
| Zdroj: | Memórias do Instituto Oswaldo Cruz Memórias do Instituto Oswaldo Cruz., Vol 113, Iss 1, Pp 38-44 (2017) Memórias do Instituto Oswaldo Cruz, Issue: ahead, Published: 27 NOV 2017 |
| ISSN: | 1678-8060 0074-0276 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|