Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods^1,2,3,4 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.

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。