title: Viral metagenome in river water Jiangsu in China
design: During 2015,  river water samples were collected from Jiangsu in China. These samples were collected as part of a Virome Project of China which aimed on discovery of emerging viruses water samples. Five hundred microliters of concentrated water 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 a mixture of DNases (Turbo DNase from Ambion, Baseline-ZERO from Epicentre, and benzonase from Novagen) and RNase (Fermentas) to digest unprotected nucleic acid at 37C for 90 min. Remaining total nucleic acid was then isolated using QIAamp MinElute Virus Spin Kit (Qiagen) according to manufacturer's protocol. Viral cDNA synthesis was performed using SuperScript III reverse transcriptase (Invitrogen) according to the manufactures instructions. The second strand of cDNA synthesis was performed by incubation of reverse transcribed (RT) products with Klenow Fragment DNA polymerase (New England Biolabs). 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 sample.