通过全长cDNA扩增及高通量测序技术获取呼肠孤病毒科病毒的全基因组序列  

Obtaining of Whole Genome Sequence of Reoviridae Viruses Through Full-length cDAN Amplification and High-throughput Sequencing Techniques

作　　者：李占鸿 宋子昂 朱建波 杨振兴 李卓然 李华春 杨恒 LI Zhan-Hong;SONG Zi-Ang;ZHU Jian-Bo;YANG Zhen-Xing;LI Zhuo-Ran;LI Hua-Chun;YANG Heng(Yunnan Tropical and Subtropical Animal Virology Laboratory,Yunnan Veterinary and Animal Science Institute,Kunming 650224,China;College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南省畜牧兽医科学院云南省热带亚热带动物病毒重点实验室,昆明650224 [2]云南农业大学动物医学院,昆明650201

出　　处：《农业生物技术学报》2021年第1期93-104,共12页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31760744);国家重点研发计划(2016YFD0500908);云南省中青年学术和技术带头人后备人才培养项目(2017HB055)。

摘　　要：获取呼肠孤病毒科(Reoviridae)病毒的全基因组序列在该科病毒的分类学和流行病学研究及诊断试剂开发等方面具有重要意义。本研究通过全长cDAN扩增(full-length amplification of cDNAs,FLAC)与二代测序(next generation sequencing, NGS)技术,获取了蓝舌病病毒(Bluetongue virus, BTV)、流行性出血病病毒(Epizootic haemorrhagic disease virus, EHDV)、帕利亚姆病毒(Palyam virus, PALV)、广西环状病毒(Guangxi orbivirus, GXOV)、西藏环状病毒(Tibet orbivirus, TIBOV)、哺乳动物正呼肠孤病毒(Mammalian orthoreovirus, MRV)、版纳病毒(Banna virus, BAV)和芒市病毒(Mangshi virus, MSV)等8种不同呼肠孤病毒科病毒的全基因组序列。本研究建立的FLAC技术,具有高度的灵敏性,无需目标病毒的任何基因序列信息与引物设计,可在1个反转录PCR (reverse transcription-PCR, RT-PCR)反应中扩增不同种属呼肠孤病毒科病毒完整的基因组DNA。扩增8种病毒的基因组大小在18 266至23 605 bp之间,基因节段数目为10或12节段。将扩增病毒的全基因组PCR产物进行NGS测序与从头组装(de novo)拼接,可在1周内获取病毒的全基因组序列。不同毒株测序产生的数据量在1.6~1.9 Gb之间,用于病毒基因组拼接的reads数(Q>30)在293 016 800至423 210 600之间,病毒基因组中每个碱基位点的测序深度在6 000至20 000之间。呼肠孤病毒科病毒全基因组扩增与高通量测序技术的建立,为快速准确地获取呼肠孤病毒科病毒的全基因组序列,开展病毒进化与变异、诊断试剂开发与流行病学等方面的研究提供了技术保障。It is vital to obtain the viral genome sequence for taxonomy, diagnostic reagent development and epidemiology studies of Reoviridae viruses. In this study, the whole genome sequences of Reoviridae viruses belonging to 8 different species were acquired through full-length cDAN amplification(FLAC) and next generation sequencing(NGS) techniques, including Bluetongue virus(BTV), Epizootic haemorrhagic disease virus(EHDV), Palyam virus(PALV), Guangxi orbivirus(GXOV), Tibet orbivirus(TIBOV), Mammalian orthoreovirus(MRV), Banna virus(BAV) and Mangshi virus(MSV). The FLAC technique established by this study showed highly sensitive and could amplify the complete genomic DNA of different species of Reoviridae viruses through one RT-PCR reaction, without any sequence information or primer designing for target viruses. The genome size of the viruses sequenced in this study ranged from 18 266 to 23 605 bp with 10 or 12 genomic segments, which were obtained in a week through NGS sequencing and de novo assembling. The amount of data generated by NGS for different Reoviridae virus strains ranged from 1.6 to 1.9 GB, with the number of reads(Q>30) used for assembling virus genome ranged from 293 016 800 to 423 210 600 and the sequencing depth for each base site in the viral genome ranged from 6 000 to 20 000. The establishment of the FLAC and NGS techniques could provide technical guarantee for rapid and accurate acquisition of the whole genome sequence of Reoviridae,which would promote the studies on evolution, diagnostic reagents development and epidemiology of Reoviridae viruses.

关 键 词：呼肠病毒科病毒 全长cDNA扩增(FLAC) 二代测序(NGS) 全基因组测序 

分 类 号：S852.659.4[农业科学—基础兽医学]