Illumina NovaSeq 6000 paired end sequencing; Sequencing of HSV-1 genomic DNA to identify genomic variation resulting from selective pressure imposed by mutation of the PP1 binding site in the pUL21 gene
Sequencing of HSV-1 genomic DNA to identify genomic variation resulting from selective pressure imposed by mutation of the PP1 binding site in the pUL21 gene
All HSV-1 strain KOS viruses were reconstituted from a bacterial artificial chromosome and mutated viruses were generated using the two-step Red recombination method. Vero cells were transfected with the recombinant BAC DNA together with pGS403 encoding Cre recombinase (to excise the BAC cassette) using TransIt-LT1. After 3 days the cells were sonicated and titrated on Vero cell monolayers. The subsequent stocks (P1 - P3) were generated by infecting cells at MOI of 0.1 until cytopathic effect was evident (approximately 4-6 days) before harvesting virus by sonication. HSV-1 genomic DNA was prepared for next-generation sequencing from partially purified nucleocapsids. 2× T150 flasks of ~70% confluent Vero cells (1.2 × 108 cells) were infected at an MOI of 0.1 and incubated at 37°C in a humidified 5% CO2 atmosphere until ~80% of cells showed cytopathic effect (4–6 days). Cells were harvested by scraping into medium, pelleted by centrifugation and lysed by resuspending in 5 mL of TNE buffer (50 mM Tris pH 7.5, 100 mM NaCl, 10 mM EDTA). Lipid envelopes were removed by adding an equal volume of Vertrel XF and mixing by inversion. The aqueous and hydrophobic phases were separated by centrifugation at 1500 g for 5 min and the aqueous phase (supernatant) was re-extracted with an additional 5 mL of Vertrel XF, mixed by inversion and separated by centrifugation. Approximately 2.8 mL of the aqueous (supernatant) phase was layered over a two-step glycerol gradient (1.3 mL of 45% (v/v) and 1.3 mL of 5% (v/v) glycerol in TNE buffer) in 5 mL thin-walled Ultraclear centrifuge tubes. Viral nucleocapsids were pelleted by centrifugation at 23,000 rpm in an SW 55 Ti rotor for 1 h at 4°C. The glycerol was removed and the pellet was drained by inverting the tube before resuspending the resultant partially-purified nucleocapsids in 400 µL sterile PBS. DNA was extracted from the enriched viral nucleocapsids using the Isolate II genomic DNA kit. DNA integrity was confirmed by agarose gel electrophoresis. Genomic DNA libraries were prepared using the NexteraXT Library Prep Kit. DNA quantification and library preparation was carried out on a Hamilton Microlab STAR automated liquid handling system.