Viral metagenomics of pig tissues

For viral metagenomic analysis, 94 different tissues specimens were collected from 94 diseased pigs from Anhui province during Sep 2017 to Sep 2018. The tissue samples of each pig were pooled into the same pool, a total of 94 pools were generated. Those pools were individually homogenized with a mortar and pestle, resuspended in 1 mL DPBS, and then frozen and thawed rapidly three times on dry-ice. The supernatants were then collected after centrifugation (10 min, 15,000 g). Supernatants were filtered through a 0.45-mm filter (Millipore) to remove eukaryotic- and bacterial cell-sized particles, and 200 uL of supernatant from each pool was then subjected to a mixture of nuclease enzymes to reduce the concentration of free (non-viral encapsidated) nucleic acids. Remaining total nucleic acid was then isolated using QIAamp MinElute Virus Spin Kit according to manufacturer's protocol. 94 libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the HiSeq Illumina platform with 250 bases paired ends with dual barcoding for each pool.