canine metagenome Genome sequencing

For viral metagenomic analysis,from 2020 to 2021, 780 fecal samples of healthy dogs, 170 fecal samples of diseased dogs were collected from Yuzhu Tibetan Autonomous Prefecture of Qinghai Province, China and 840 fecal samples of healthy dogs and 180 fecal samples of diseased dogs were collected from Gulo Tibetan Autonomous Prefecture of Qinghai Province, China, for a total of 1970 samples. Samples were randomized faccording to region and level of health.The fecal samples 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. One hundred and ninety-seven libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the MiSeq Illumina platform with 250 bases paired ends with dual barcoding for each pool.