Obtaining of Whole Genome Sequence of Reoviridae Viruses Through Full-length cDAN Amplification and High-throughput Sequencing Techniques
LI Zhan-Hong1,*, SONG Zi-Ang2 *, ZHU Jian-Bo1, YANG Zhen-Xing1, LI Zhuo-Ran1, LI Hua-Chun1, YANG Heng1,**
1 Yunnan Tropical and Subtropical Animal Virology Laboratory, Yunnan Veterinary and Animal Science Institute, Kunming 650224, China; 2 College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China
Abstract：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.
李占鸿, 宋子昂, 朱建波, 杨振兴, 李卓然, 李华春, 杨恒. 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[J]. 农业生物技术学报, 2021, 29(1): 93-104.
LI Zhan-Hong, SONG Zi-Ang, ZHU Jian-Bo, YANG Zhen-Xing, LI Zhuo-Ran, LI Hua-Chun, YANG Heng. Obtaining of Whole Genome Sequence of Reoviridae Viruses Through Full-length cDAN Amplification and High-throughput Sequencing Techniques. 农业生物技术学报, 2021, 29(1): 93-104.
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