Human papillomavirus virus isolate from vaginal secretions of pregnant women Metagenome

During 2015-2017, 60 vaginal secretion samples were collected from pregnant women with colpitis (n=40) and normal pregnant women (n=20) in Zhenjiang in China. Samples were resuspended in 10 volumes of phosphate-buffered saline (PBS) and vigorously vortexed for 5 min. Supernatant were then collected and pooled into 6 sample pools (each containing 10 samples) according to health status. Five hundred microliters of each pool was collected after centrifugation (10 min, 15,000 g) and 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 37 Celsius degree 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 pool.