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通过直接RNA测序验证多样化和以前无法追踪的仙台病毒复制病毒基因组

Sarah E Pye, Emna Achouri, Yanling Yang

bioRxiv : the preprint server for biology

|April 16, 2025

概括

直接RNA测序 (DRS) 和一个新的分析工具 (LoCA) 实现了复制病毒基因组 (cbVGs) 的全长测序. 这克服了基于PCR的方法的局限性,为cbVG多样性和病毒感染中的角色提供了更清晰的画面.

科学领域:

病毒学病毒学
分子生物学分子生物学
生物信息学是一种生物信息学.

背景情况:

复制病毒基因组 (cbVGs) 是截断的病毒RNA分子,具有互补的末端,在负感RNA病毒复制过程中产生.
cbVGs通过干扰复制,激活抗病毒反应和抑制蛋白质翻译,在病毒感染结果中发挥关键作用.
目前用于研究cbVG的方法,如基于PCR的方法,受到偏向较短序列,错误的可能性和无法捕获全长基因组的限制.

研究的目的:

优化直接RNA测序 (DRS) 以获得cbVGs的原生全长序列.
开发一个强大的生物信息学工具,用于从长期读取的测序数据中识别cbVGs.
分析仙台病毒库存中的cbVGs的多样性,并验证新的测序和分析方法.

主要方法:

直接RNA测序 (DRS) 协议的优化,用于测序来自仙台病毒的全长cbVGs.
设计和实施基于BLAST的分析脚本,长读cbVG分析 (LoCA),用于在长读测序数据中检测cbVG.
分析来自多个仙台病毒库存的DRS数据,包括优化对主导cbVG捕获的寡核酸长度,并测试低RNA输入量.

主要成果:

优化的DRS协议成功地以高完整性对全长cbVGs进行测序,相比之前的建议 (1000ng),需要显著减少RNA输入 (17.6-50ng).
该LoCA脚本有效地从DRS数据中识别cbVGs,在不同的仙台病毒库存中展示了实用性.
成功测序了各种cbVG物种,包括具有在参考基因组开始或接近基因组开始的突破位置的新物种,并确定了捕获主导cbVGs的最佳条件.

结论:

直接RNA测序 (DRS) 与LoCA分析工具相结合,提供了一种强大且易于使用的全长cbVG测序方法,克服了现有技术的局限性.
这种优化的方法使我们能够更全面地了解cbVG多样性及其对病毒感染的影响.
这些发现有助于对病毒库存进行常规测序,以评估cbVGs在不同病毒感染中的作用.

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