human viral metagenome Metagenome

For viral metagenomic analysis, 90 Fecal specimens were collected from 45 neonates who were born in the Affiliated Hospital of Jiangsu University and 45 healthy children who were examined at the same hospital from Sep. 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. Then the 90 supernatants were randomly pooled into 10 specimen pools, each including 9 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. Ten 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.