Virome Characterization via Ion Torrent

Sample preparation and sequencing was done following protocols optimized for virome characterization using the Ion Torrent platform (Thermo Fisher Scientific, Waltham, MA, USA) (Vibin et al., 2018 (link); Conceição-Neto et al., 2015 (link)). Prior to library preparation, mucosal samples were purified and enriched to remove host cells and free nucleic acids as follows. Samples were first low-speed centrifuged at 5,200 g for 10 min to remove cell debris, then filtered using centrifugation through a 0.45 µm cellulose acetate filter (Spin-X Centrifuge tube filter, cat#8162; Corning Inc., Corning, NY, USA) at 13,000 g for 1 min. Subsequently, to remove free nucleic acids, the filtrate was treated with Pierce Universal Nuclease (cat#88702; Thermo Fisher Scientific, Waltham, MA, USA) at the rate of 10 units of enzyme per 1 ml of solution. Finally, samples were concentrated using a Pierce™ PES protein concentrator (cat#88503), allowing concentration of proteins at 100 K molecular weight cut-off (Thermo Fisher Scientific, Waltham, MA, USA) at 13,000 g to a final volume of 150 µl/sample.