Computational protocol: The full genome sequences of 8 equine herpesvirus type 4 isolates from horses in Japan

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Protocol publication

[…] Genome sequences of TH20p, 05-I-202 and 12-I-203 were read by the next generation sequencer (NGS) GS Junior (Roche Diagnostics K. K., Tokyo, Japan), and those of 83-MB, 91c1, 01-10-1 03-VR and 11-10 were read by the NGS MiSeq (Illumina lnc., Tokyo, Japan) according to the manufacturer’s protocol. The complete genome sequences were assembled by reference sequence mapping with Bowtie 2 [] using EHV-4 strain NS80567 sequence (GenBank accession number AF030027) or TH20p sequence as the reference and editing with Consed [] and SnapGene software (GSL Biotech; available at The full genome sequence of TH20p was determined by mapping against the NS80567 sequence. The genome sequences of the other 7 Japanese isolates were determined by mapping against TH20p as a reference sequence. Because ORFs 24 and 71 included large regions of direct repeat sequences, these regions were amplified by PCR and further analyzed by Sanger sequencing. The primers used to amplify the ORFs 24 and 71 were ORF24-F (5′-gctttccaaaccttggcgtccatcgatacg-3′), ORF24-R (5′-ccgcgcggtttatctgtagatcatattcaagttc-3′), ORF71-F (5′-ctacatcaacctcggtgtc-3′) and ORF71-R (5′-cctgtttgatccaaccgacc-3′). Direct repeat sequences were analyzed by Tandem Repeat Finder [] and XSTREAM [] and removed from sequences before multiple alignments. The locations of removal are indicated in . The whole genome, UL, US and IR of EHV-4s including sequences determined in the present study, and EHV-4 reference strain NS80567 and 14 sequences of Australian isolates [] were multiple aligned by MAFFT version7 [] and AliView 1.18 []. Phylogenetic trees were constructed by neighbor-joining method using the Molecular Evolutionary Genetic Analysis (MEGA7) software [] based on the nucleotide sequences excluding direct repeat sequences. Each ORF’s amino acid (AA) sequence was analyzed as the same as nucleic acid sequences. The AA differences were counted using AliView 1.18 manually. […]

Pipeline specifications

Software tools Bowtie, Consed, XSTREAM, MAFFT, AliView, MEGA
Application Phylogenetics
Organisms Equus caballus, Equid alphaherpesvirus 4
Diseases Respiratory Insufficiency
Chemicals Amino Acids