Computational protocol: The Emergence of Rotavirus G12 and the Prevalence of Enteric Viruses in Hospitalized Pediatric Diarrheal Patients in Southern Vietnam

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Protocol publication

[…] PCR amplicons from successful VP7 and VP4 PCR amplifications were DNA sequenced using the amplification primers. PCR amplicons were purified using the QIAquick PCR purification kit (QIAGEN, Germany), and DNA concentrations were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, United Kingdom). Direct DNA sequencing was performed using the BigDye Terminator Cycle Sequencing kit (Applied Biosystems) according to the manufacturer's recommendations. All DNA sequences were generated using an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems), and the resulting DNA sequences were assembled using DNA Baser Sequence Assembler v3.0.17 (Heracle Biosoft, Pitesti, Romania).The resulting VP4 and VP7 sequences (Genbank accession numbers: VP4, FR820957–FR821065; VP7, FR822209–FR822321) were compared with other corresponding genotype sequences using BLASTn (https://blast.ncbi.nlm.nih.gov/ Blast.cgi) for genotype determination. Coding sequences were manually aligned using Se-AL v2.0a11 (http://tree.bio.ed.ac.uk/software/), and additional sequences were trimmed to correspond with our sequences to maximize sequence homology. Maximum likelihood trees for each of the genotypes were inferred using RAxML v 7.0.426 employing the general time-reversible model of nucleotide substitution with a γ-distribution of among-site rate variation (GTR+Γ), which was determined using jModelTest. One thousand bootstrap replicates were used as implemented in a rapid bootstrap algorithm available in RAxML. The resulting trees were visualized in FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/), and genetic distances were estimated using HyPhy v2.0 (http://www.datam0nk3y.org/hyphy/doku.php). All statistical analyses were performed in R version 2.9.0 (The R foundation for Statistical Computing); P values < 0.05 were considered statistically significant. […]

Pipeline specifications

Software tools DNA Baser Sequence Assembler, BLASTN, Se-Al, RAxML, jModelTest, FigTree, HyPhy
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Homo sapiens
Diseases Infection