title: Viral metagenomics reveals many viruses in the faces samples of healthy raccoon dogs:lib18
design: In 2014, 30 fecal samples of healthy raccoon dog were collected from a breeding base in Jilin Province, China. Samples were shipped in dry ice and re-suspended in ten volumes of phosphate-buffered saline (PBS) and vigorously vortexed for 10 min, then centrifuged at 12,000 rpm for 10 min. Finally every fecal supernatant was collected in a new 1.5 ml centrifuge tubes. Three pools contained 10 fecal suspensions were randomly generated. Total 500 ul fecal suspension equal from 10 feces samples was mixed together. The supernatants were then collected after centrifugation (10 min, 15,000g). 500 l of each supernatant was filtered through a 0.45-m filter (Millipore) to remove eukaryotic and bacterial cell-sized particles. The filtrates enriched in viral particles were treated with DNase and RNase to digest unprotected nucleic acid at 37C for 60 min. Remaining total nucleic acid was then isolated using QIAamp MinElute Virus Spin Kit (Qiagen) according to manufacturer's protocol. Three libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the Illumina Miseq platform with 250 bases paired ends with dual barcoding for each pool. For bioinformatics analysis, paired-end reads of 250 bp generated by MiSeq were debarcoded using vendor software from Illumina.