title: OrNV-S2A_AgR
design: Infected cells were detached, collected by centrifugation, resuspended in water, and lysed by sonication, 5 rounds of 20 seconds (MSE Sanyo Soniprep 150, 6.5 m amplitude, 23 KHz). Supernatant from OrNV isolates PV505, V23B, X2B and PNG was treated with DNaseI (Thermo Fisher Scientific, New Zealand) to remove host cell DNA. OrNV-S2A and Dug42 were not treated with DNaseI. All isolates were treated with RNaseA and Proteinase K (New England Biolabs, New Zealand). Viral DNA was extracted with phenol/chloroform/isoamyl alcohol (Sigma-Aldrich, Australia), chloroform/isoamyl alcohol, precipitated with 100% ethanol and 3M sodium acetate (pH 5.2), washed with 70% ethanol, and resuspended in TE buffer. Nanopore libraries for OrNV-PV505, -V23B, -X2B and -PNG were prepared using PCR barcoding gDNA protocol (EXP-PBC001 and SQK-LSK110; ONT). Libraries for OrNV-S2A and -Dug42 were prepared using the genomic DNA by ligation sequencing protocol (SQK-LSK110; ONT). All libraries were prepared without a DNA shearing step.