5 μm centrifuge filters

Extracts of total nucleic acid were obtained by using the QIAamp DNA mini kit (Qiagen) according to manufacturer’s instructions with the addition of 10 μg linear acrylamide as carrier (Applied Biosystems) at the ethanol precipitation step. QIAamp DNA mini kit was used for both DNA and RNA extractions, as RNA yields equalled that of commonly used RNA columns (RNeasy mini kit). Virion enrichment was performed by centrifugation for 2 minutes at 800 × g to remove tissue debris, and the supernatants were subsequently filtered through 5 μm centrifuge filters (Millipore). In addition to virus discovery, the methods were also designed for inclusion of bacteria, and thus a filter pore size of 5μm was chosen for the filtration. The filtrates were nuclease treated to remove unprotected nucleic acids using 7 μl TURBO DNase (2U/μl) (Ambion), 6 μl Baseline-ZERO DNase (1U/μl) (Epicentre), 8 μl RNase Cocktail Enzyme Mix (Ambion), and 20 μl 10× TURBO DNase buffer in a final volume of 200 μl, and incubated at 37°C for two hours. Viral nucleic acids were subsequently extracted using Roche High Pure Viral RNA kit (Roche).

Next-generation sequencing (NGS) was used to sequence the poxvirus genomes. Virion enrichment was performed by centrifugation for 2 min at 800 × g to remove tissue debris, and the supernatants were subsequently filtered through 5 μm centrifuge filters (Millipore) [43 (link)]. The filtrates were nuclease treated to remove unprotected nucleic acids using 8 μL RNase Cocktail Enzyme Mix (Invitrogen). Viral nucleic acids were subsequently extracted using QIAamp DNA mini (Qiagen). The genomic libraries were prepared with an insert size of 150 paired-end. DNA sequencing (NGS) was performed on a HiSeq4000 sequencing platform (Illumina) by Novogene, China.