human viral metagenome Genome sequencing

For viral metagenomic analysis, 50 blood specimens, 50 oral swab specimens and 50 Fecal specimens were collected from newborns who were admitted to the newborn department of the Affiliated Hospital of Jiangsu University from Sep 2018 to Dec 2018. Each specimen was added 200 to 500 ul DPBS. And then each specimen was shaken for 5 minutes and repeated it three times. The supernatants were collected after centrifugation (10 min, 15,000 × g). Then the 150 supernatants were randomly pooled into 15 specimen pools, each including 10 specimens. 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. Fifteen libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Illumina sequencing platform with 250 bases paired ends with dual barcoding for each pool.