Viral Genome Sequencing from EPC Cell Culture

Inoculation of the ECIV isolate onto EPC cells in four 175 cm² flasks at a high multiplicity of infection provided third-passage material harvested after 21 days post-infection when CPE was extensive. Cell culture supernatant was clarified at 5520× g for 20 min at 4 °C. The pelleted virus was obtained by centrifugation of the clarified supernatant at 100,000× g for 1.25 h at 4 °C. The viral pellet was resuspended in 360 μL of animal tissue lysis (ATL) buffer prior to extraction of viral genomic DNA using a DNeasy Blood and Tissue Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. A DNA library was generated using a Nextera XT DNA Kit, and sequencing was performed using a V3 chemistry 600 cycle Kit on a MiSeq sequencer (Illumina, Germantown, MD, USA). De novo assembly of the paired-end reads was performed in SPAdes 3.5.0 genome assembly algorithm [22 (link)]. The quality of the genome assembly was verified by mapping the reads back to the consensus sequence in Bowtie 2 2.1.0 [23 (link)] and visually inspecting the alignment in Tablet 1.14.10.20 [24 (link)].