相关实验视频
Updated: Jul 1, 2025
10:34
Ultra-long Read Sequencing for Whole Genomic DNA Analysis
Published on: March 15, 2019
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一个长时间阅读的测序策略,在相邻的片段上重叠的链接器 (OLAF-Seq),用于有针对性的重新测序和丰富
Lahari Uppuluri1, Christina Huan Shi2, Dharma Varapula1
1School of Biomedical Engineering, Science and Health Systems, Drexel University, Philadelphia, PA, 19104, USA.
Scientific reports
|March 6, 2024
概括
我们开发了OLAF-Seq,这是一种使用CRISPR-Cas9连接DNA片段的新方法,使得目标基因组区域的高效长读测序能够通过改进的组装和分阶段进行. 这种方法可以为复杂的基因组分析提供深度测序.
科学领域:
- 基因组学就是基因组学.
- 分子生物学分子生物学
- 生物技术是生物技术.
背景情况:
- 长读测序为基因组组装和结构变异检测提供了优势.
- 针对性测序方法对于对特定基因组区域的成本效益分析至关重要.
- 保持DNA片段之间的物理联系对于精确的基因组重建至关重要.
研究的目的:
- 引入OLAF-Seq,这是一个用于构建长时间阅读序列库的新策略.
- 为了实现大型基因组区域的有针对性的丰富和深度测序,同时保留链接信息.
- 为了证明OLAF-Seq对哈普罗型分相和基因组组合的有用性.
主要方法:
- 利用CRISPR-Cas9尼克酶和多个sgRNA用于长DNA分子 (>300kb) 的有针对性,非随机的碎片化.
- 生成的图书馆大小的碎片 (大约. 20 kbp) 保持相邻段之间的物理链接信息 (高达1000 bp).
- 应用适配器结合用于长读测序平台的向DNA分子的优先丰富 (PacBio,牛津纳米孔).
主要成果:
- 使用菌体DNA和测序100个与癌症相关的人类基因 (外子和内子) 证明了OLAF-Seq的有效性.
- 实现了有针对性的测序,总覆盖率明显降低,利用链接区域进行组装和分阶段.
- 当设计的链接器含有异合体遗传变异时,成功建立了长单元类型.
结论:
- OLAF-Seq是一个多功能,具有成本效益的测序策略,适用于PacBio和牛津纳米孔平台.
- 该方法促进了对众多基因组区域的有针对性的丰富,并产生长,无间隙的连接.
- OLAF-Seq为复杂的遗传变异的基因组组,分相和分析提供了宝贵的信息.
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