Parvovirus dark matter in the feces of wild birds. viral metagenome

From 2016 to 2018, to investigate the virome of wild birds, more than 4,000 cloacal swabs were collected from 8 different provinces in China. All samples were collected by disposable materials. Each sample was re-suspended in ten volumes of phosphate-buffered saline and vigorously vortexed for 5 min. The supernatants were then collected after centrifugation. 500 ul of each supernatant was filtered through a 0.45 um filter 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 for 60 min. Remaining total nucleic acid was then isolated using QIAamp MinElute Virus Spin Kit according to manufacturer protocol. More than 300 libraries (each library contained pooled samples) were then constructed using Nextera XT DNA Sample Preparation Kit and sequenced using the Illumina Hiseq 2500 or NovaSeq 6000 platform with 250 bases paired ends with dual barcoding for each library. For bioinformatics analysis, paired end reads of 250 bp generated by Illumina MiSeq or NovaSeq platform were debarcoded using vendor software from Illumina