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Pipeline publication

[…] uration (1 minute at 95 °C), annealing (1 minute at 60.8 °C) and elongation (2 minutes at 72 °C)., A 380-bp product from the viral DNA polymerase gene was amplified and digested with SalI as described previously [], using template DNA from samples (n = 18) that had not been previously assigned to N/D752 variants. The viruses were assigned to N/D752 variants based on restriction fragment length polymorphism (RFLP) of the digested product., PCR products of the expected sizes from both ORF30 and ORF68 PCR were sequenced using BigDye® Terminator version 3.1 (Applied Biosystems) on a 3730xl DNA Analyzer (Applied Biosystems) at Genomed (Warsaw, Poland). The obtained sequences were assembled using BioEdit software (version 7.2.5). Alignment and comparison of the nucleotide sequences were carried out using ClustalW in MEGA version 5.0.5. []., A total of 37 EHV-1 ORF68 PCR products that generated good-quality sequence data were used for phylogenetic analysis (the sequence from PL_2015_I was of insufficient quality). Sequences were aligned in Geneious v 9.1.3, and ambiguous base calls where resolved by reference to chromatographs. For each sequence, a consensus sequence was generated from the forward and reverse sequencing products. Additional EHV-1 sequences (n = 178) originating from various countries (including one Polish sequence) were obtained from the National Centre for Biotechnology Information (NCBI) database. Selected sequences had an ORF68 annotation, were at least 500 bp in length, and were not the only representative from a given country. An alignment of all 215 EHV-1 ORF68 sequences (464 bp) was generated in Geneious v 9.1.3, and exported in nexus file format for downstream analysis., Population structure analysis w […]

Pipeline specifications

Software tools BioEdit, Clustal W, MEGA, Geneious