Super-cutting-edge Grand and Advanced Research (SUGAR) Program, Japan Agency for Marine Science and Technology (JAMSTEC).
Hakubi Center for Advanced Research, Kyoto University.
Microbes Environ. 2021;36(2). doi: 10.1264/jsme2.ME20152.
Fragmented and primer ligated dsRNA sequencing (FLDS) is a sequencing method applicable to long double-stranded RNA (dsRNA) that enables the complete genome sequencing of both double- and single-stranded RNA viruses. However, the application of this method on a low amount of dsRNA has been hindered by adaptor dimer formation during cDNA amplification and sequence library preparation. We herein developed FLDS ver. 3 by optimizing the terminal modification of an oligonucleotide adaptor and the conditions of adaptor ligation. We also examined the concentration of Mg in the PCR reaction for cDNA amplification and the purification method of amplified cDNA. Fine sequence reads were successfully obtained from metagenomic shotgun sequencing libraries constructed from 10 and 100 pg dsRNA, and these libraries exhibited weaker detection sensitivity for low-abundance dsRNAs (viral genomes and genome segments) than that constructed from 1 ng of dsRNA. We also report the utility of capillary electrophoresis for dsRNA quantification. The FLDS ver. 3 package expands the frontiers of our knowledge in RNA virus diversity and evolution.
片段化和引物连接的双链 RNA 测序 (FLDS) 是一种适用于长双链 RNA (dsRNA) 的测序方法,可实现双链和单链 RNA 病毒的全基因组测序。然而,由于 cDNA 扩增和文库制备过程中接头二聚体的形成,该方法在低量 dsRNA 上的应用受到了限制。我们通过优化寡核苷酸接头的末端修饰和接头连接条件,开发了 FLDS ver.3。我们还研究了 cDNA 扩增 PCR 反应中 Mg 的浓度和扩增 cDNA 的纯化方法。从 10 和 100 pg dsRNA 构建的宏基因组鸟枪法测序文库中成功获得了精细的序列读数,与从 1ng dsRNA 构建的文库相比,这些文库对低丰度 dsRNA(病毒基因组和基因组片段)的检测灵敏度较弱。我们还报告了毛细管电泳用于 dsRNA 定量的实用性。FLDS ver.3 包扩展了我们对 RNA 病毒多样性和进化的认识。
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