0.45 μm pore size sterile disk filters

Conventional virus enrichment methods commonly used in metagenomic sequencing-based virus discovery include filtration and pre-extraction nuclease treatments, often combined with postextraction DNase I and/or depletion of ribosomal rRNA sequences. Briefly, samples (100 to 300 µl) were filtered through 0.45-μm-pore-size sterile disk filters (Merck/Millipore, Billerica, MA, USA) to enrich for viruses over cells or bacteria. The flowthrough was treated with 1 μl RNase A (10 mg/ml; Thermo, Fisher, Waltham, MA, USA) for 15 min at room temperature, followed by a cocktail of 8 U Turbo DNase (Thermo, Fisher), 250 U Benzonase (Merck/Millipore), and 10 mM MgCl₂ for 45 min at room temperature to digest non-particle-protected NAs. Protected NAs, such as those in viral particles, were extracted by easyMAG (bioMérieux) or AllPrep kits (Qiagen). Postextraction digestion by DNase I (2 U/µg DNA for 15 min at 37^oC; Thermo, Fisher) was added in some instances to digest chromosomal DNA (cellular and bacterial), but it will also digest viral DNA (e.g., HHV-1 DNA), whereas mRNA transcripts generated from actively replicating cellular virus would be maintained. Depletion of nondesired host mRNA sequences was achieved using RiboZero magnetic kits (Illumina, San Diego, CA, USA). Enriched preparations were subjected to reverse transcription and sequence library preparation.