Computational protocol: Isolation of Infective Zika Virus from Urine and Saliva of Patients in Brazil

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Protocol publication

[…] A total of 17,413,830 reads was generated for Rio-U1 sample and 21,734,486 for Rio-S1 sample. Related reads to Chlorocebus sabaeus have been filtered using Bowtie2 and Samtools, remaining 12,614,062 reads of Rio-U1 and 12,943,134 of Rio-S1. Both genomes were assembled using Ray 2.20 (k = 31). The completed genome of Rio-U1 has 10,795bp (Accession number KU926309) and Rio-S1 has 10,805bp (Accession number KU926310). Gene prediction was performed by GenemarkS 4.17. Mature peptides were identified by blastp against the protein annotated in reference sequence NC_012532. [...] Nucleotide sequences encoding the precursor polyprotein of 39 ZIKV strains and 1 of DENV 4 were aligned using the Clustal W []. Evolutionary analysis was performed as described elsewhere []. Phylogenetic studies were carried out using the Maximum Likelihood method based on the General Time Reversible model [] of the MEGA7 software []. Evolutionary history of these sequences was represented by bootstrap consensus tree (from 1000 replicates), in a traditional branch style. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. Initial trees for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories; +G, parameter = 0.9645). The rate variation model allowed some sites to be evolutionarily invariable ([+I], 37.8665% sites). The analysis involved 40 nucleotide sequences. All positions with less than 95% site coverage were eliminated. There were 10247 positions in the final dataset. Timetree was inferred by Reltime method [] from MEGA, using GTR (G+I, 5 categories), partial deletion with site coverage cutoff of 95%. […]

Pipeline specifications

Software tools Bowtie2, SAMtools, GeneMarkS, BLASTP, Clustal W, MEGA
Applications Genome annotation, Phylogenetics
Organisms Zika virus, Homo sapiens, Human poliovirus 1 Mahoney
Diseases Exanthema, Microcephaly, HIV Infections, Guillain-Barre Syndrome