Viral metagenomics of fecal samples from lizards in the Qinghai-Tibetan Plateau: Blank Control
In Jul. 2020, to investigate the virome of two lizard species in the Qinghai-Tibetan Plateau, 81 lizard fecal samples were collected from five sample sites in the Plateau. All samples were collected by disposable materials. Fecal samples were pooled into 6 libraries according to lizard species and sampling sites. As a blank control, sterile ddH2O was simultaneously prepared and further processed in the same condition. Samples were re-suspended in ten volumes of phosphate-buffered saline (PBS) and vigorously vortexed for 5 min, following frozen and thawed three times on dry ice. The supernatants were then collected after centrifugation (10 min, 15,000g). Totally, 500 uL of each supernatant was filtered through a 0.45 um 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. Six libraries were then constructed using Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced using the NovaSeq6000 platform with 250 bases paired ends and dual barcoding for each pool. For bioinformatics analysis, paired-end reads of 250 bp generated by NovaSeq were debarcoded using vendor software from Illumina.