Bird Metagenome

For viral metagenomic analysis, in 2018, cloacal swabs of 58 birds of 14 species from 3 orders (Gruiformes, Galliformes, Psittaciformes) were collected from a zoo in Nanjing city in Jiangsu province. The 58 cloacal swabs were randomly pooled into 4 specimen pools, each including 14 or 15 cloacal swabs. The cloacal swabs pools were individually 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. Eleven 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.