Cressdnaviruses of Cynopterus bat

This study used metagenomic to characterize cressdnaviruses in a rectal swab sample of a GGreater short-nosed fruit bat (Cynopterus sphinx). Two aliquots of the sample in question were retrieved from the -80 degC freezer and centrifuged at 1800 x g and 4 degC, for 15 min. Eukaryotic and bacterial cell debris was removed by passing the supernatants through a filter with 0.45 um pores. The first aliquot (the untreated sample) was used for nucleic acid extracted with the RNA Easy Fast Tissue/Cell kit (TIANGEN). In the second aliquot (treated sample), the unprotected nucleic acids were digested with a cocktail of enzymes - 15 U Turbo DNase, 20 U Benzonase, 20 U RNase One, in 10x Turbo DNase buffer - and 30 mM EDTA was then added and incubated with the mixture for 30 minutes to stop the enzymatic reaction (120,121). The nucleic acids obtained from the two sample aliquots were reverse-transcribed and the libraries were prepared using NEBNext Ultra II library preparation kit, in accordance with the protocol recommended by the manufacturer (Illumina) for use with FFPE RNA. The libraries were pair end-sequenced on an Illumina Novogene 6000 device based in Nanjing, China.

本研究采用宏基因组学（metagenomic）方法，对一只大短鼻果蝠（Cynopterus sphinx）的直肠拭子样本中的环状单链DNA病毒（cressdnaviruses）进行特征解析。研究人员从-80℃冰箱中取出该样本的两份分装冻存管，于4℃、1800×g条件下离心15分钟；将上清液通过0.45μm孔径的滤膜过滤，以去除真核与细菌细胞碎片。第一份分装样本（未处理组）采用RNA Easy Fast Tissue/Cell试剂盒（TIANGEN）提取核酸。第二份分装样本（处理组）中，未受保护的核酸采用酶混合液进行消化——该混合液包含15 U Turbo DNase、20 U Benzonase、20 U RNase One，溶于10×Turbo DNase缓冲液中；随后加入30 mM EDTA并与反应体系孵育30分钟以终止酶促反应(120,121)。提取自两份分装样本的核酸均进行逆转录，随后参照Illumina官方推荐的适配福尔马林固定石蜡包埋（FFPE）RNA的建库流程，使用NEBNext Ultra II文库制备试剂盒构建测序文库。所构建的测序文库于中国南京的Illumina Novogene 6000测序平台上完成双端测序。