多种天然形式RNA病毒的快速测序
Rapid Sequencing of Multiple RNA Viruses in Their Native Form.
作者信息
Wongsurawat Thidathip, Jenjaroenpun Piroon, Taylor Mariah K, Lee Jasper, Tolardo Aline Lavado, Parvathareddy Jyothi, Kandel Sangam, Wadley Taylor D, Kaewnapan Bualan, Athipanyasilp Niracha, Skidmore Andrew, Chung Donghoon, Chaimayo Chutikarn, Whitt Michael, Kantakamalakul Wannee, Sutthent Ruengpung, Horthongkham Navin, Ussery David W, Jonsson Colleen B, Nookaew Intawat
机构信息
Department of Biomedical Informatics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, TN, United States.
出版信息
Front Microbiol. 2019 Feb 25;10:260. doi: 10.3389/fmicb.2019.00260. eCollection 2019.
Long-read nanopore sequencing by a MinION device offers the unique possibility to directly sequence native RNA. We combined an enzymatic poly-A tailing reaction with the native RNA sequencing to (i) sequence complex population of single-stranded (ss)RNA viruses in parallel, (ii) detect genome, subgenomic mRNA/mRNA simultaneously, (iii) detect a complex transcriptomic architecture without the need for assembly, (iv) enable real-time detection. Using this protocol, positive-ssRNA, negative-ssRNA, with/without a poly(A)-tail, segmented/non-segmented genomes were mixed and sequenced in parallel. Mapping of the generated sequences on the reference genomes showed 100% length recovery with up to 97% identity. This work provides a proof of principle and the validity of this strategy, opening up a wide range of applications to study RNA viruses.
使用MinION设备进行的长读长纳米孔测序提供了直接对天然RNA进行测序的独特可能性。我们将酶促聚腺苷酸化反应与天然RNA测序相结合，以（i）并行测序单链（ss）RNA病毒的复杂群体，（ii）同时检测基因组、亚基因组mRNA/mRNA，（iii）无需组装即可检测复杂的转录组结构，（iv）实现实时检测。使用该方案，将正链ssRNA、负链ssRNA、有/无聚（A）尾、分段/非分段基因组混合并并行测序。将生成的序列映射到参考基因组上显示，长度回收率达100%，同一性高达97%。这项工作提供了该策略的原理证明和有效性，为研究RNA病毒开辟了广泛的应用。
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