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Targeted nanopore sequencing with Cas9-guided adapter ligation
Nature Biotechnology ( IF 33.1 ) Pub Date : 2020-02-10 , DOI: 10.1038/s41587-020-0407-5
Timothy Gilpatrick 1 , Isac Lee 1 , James E Graham 2 , Etienne Raimondeau 2 , Rebecca Bowen 2 , Andrew Heron 2 , Bradley Downs 3 , Saraswati Sukumar 3 , Fritz J Sedlazeck 4 , Winston Timp 1, 5
Affiliation  

Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1,2,3,4 are limited by the loss of native modifications, short read length, high input requirements, low yield or long protocols. In the present study, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adapters for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants, structural variations and CpG methylation. We apply nCATS to four cell lines, to a cell-line-derived xenograft, and to normal and paired tumor/normal primary human breast tissue. Median sequencing coverage was 675× using a MinION flow cell and 34× using the smaller Flongle flow cell. The nCATS sequencing requires only ~3 μg of genomic DNA and can target a large number of loci in a single reaction. The method will facilitate the use of long-read sequencing in research and in the clinic.



中文翻译:


使用 Cas9 引导的接头连接进行靶向纳米孔测序



尽管测序方法最近有所改进,但仍然需要提供高测序深度和全面变异检测的测定方法。目前的方法1、2、3、4受到本机修改丢失、读长短、输入要求高、产量低或协议长的限制。在本研究中,我们描述了纳米孔 Cas9 靶向测序 (nCATS),这是一种利用 Cas9 对染色体 DNA 进行靶向切割来连接适配器以进行纳米孔测序的富集策略。我们证明 nCATS 可以同时评估单倍型解析的单核苷酸变异、结构变异和 CpG 甲基化。我们将 nCATS 应用于四种细胞系、细胞系衍生的异种移植物以及正常和配对的肿瘤/正常原代人乳腺组织。使用 MinION 流动槽时的中位测序覆盖度为 675 倍,使用较小的 Flongle 流动槽时为 34 倍。 nCATS 测序仅需要约 3 μg 基因组 DNA,并且可以在一次反应中靶向大量基因座。该方法将促进长读长测序在研究和临床中的使用。

更新日期:2020-02-10
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