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高通量纳米孔DNA测序大型插入体克隆直接从细菌殖民地

Léa Chuzel¹, Amit Sinha¹, Caileigh V Cunningham¹

¹New England Biolabs, Ipswich, Massachusetts, USA.

Applied and environmental microbiology

|May 20, 2024

概括

我们开发了一种使用纳米孔技术直接从细菌群中测序体DNA的快速方法. 这种方法与phiXXer生物信息学管道相结合,加速了功能性元基因组学研究中的基因和途径的发现.

科学领域:

转基因组学是指转基因组学.
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:

体和宇宙体是功能性元基因组研究的关键载体,因为它们具有很大的插入容量 (~30 kb).
在功能查后,对体插件的测序是识别基因或通路的关键瓶.
目前的方法需要大量的样本准备,限制了吞吐量.

研究的目的:

开发一种快速,高通量方法,直接从细菌殖民地进行体测序.
创建一个生物信息学管道,用于 de novo 组装和向量剪切的 fosmid 序列.
克服目前在功能元基因组学中的体测序技术的局限性.

主要方法:

从 lysed Escherichia coli 菌群中直接对体进行纳米孔测序,无需培养或净化.
使用phi29聚合酶进行体DNA放大.
开发phiXXer生物信息管道,用于读取组装和矢量修剪.

主要成果:

准确的 de novo 测序 fosmid 插件 (~30-40 kb) 直接从殖民地.
在单个纳米孔运行中成功对96个体进行了测序.
使用含有基因组和元基因组DNA的体库进行验证.

结论:

新方法显著加速了体测序,这是功能性元基因组发现的关键步骤.
直接从殖民地纳米孔测序绕过了繁的培养和净化步骤.
phiXXer管道使线性体插入序列的高效组装成为可能,增强了发现潜力.

关键词:

在DNA测序过程中,DNA测序福斯米德 (Fosmid) 是一种体.转基因组学是指转基因组学.这是一个纳米孔.

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