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Cas9介导的纳米孔测序技术
作者:小柯机器人 发布时间:2020/2/13 10:09:11

美国约翰•霍普金斯大学Winston Timp研究组的一项最新研究利用Cas9引导的衔接子连接建立了靶向的纳米孔测序技术。该项研究成果在线发表在2020年2月10日出版的《自然—生物技术》上。

研究人员建立了纳米孔Cas9靶向测序(nCATS)技术,其利用Cas9靶向切割染色体DNA来连接适配器进行纳米孔测序,这是一种富集策略。研究表明,nCATS可以同时在单倍型水平上检测单核苷酸突变、结构变异和CpG甲基化。研究人员将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成对的肿瘤/正常原发性人类乳房组织。MinION测序方法的平均测序覆盖率为675x,而较小的Flongle测序的测序覆盖率为34x。nCATS测序仅需要约3μg基因组DNA,并且可以在单个反应中靶向大量基因座。该方法将有助于长测序在研究和临床中的使用。

研究人员表示,尽管近年来测序方法有所改进,但仍需要提供高测序深度和全面突变的检测方法。当前的测序方法受序列修饰缺失、读取长度短、输入要求高、产率低或测序方法冗长的限制。

附:英文原文

Title: Targeted nanopore sequencing with Cas9-guided adapter ligation

Author: Timothy Gilpatrick, Isac Lee, James E. Graham, Etienne Raimondeau, Rebecca Bowen, Andrew Heron, Bradley Downs, Saraswati Sukumar, Fritz J Sedlazeck, Winston Timp

Issue&Volume: 2020-02-10

Abstract: 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 ~3μg 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.

DOI: 10.1038/s41587-020-0407-5

Source: https://www.nature.com/articles/s41587-020-0407-5

期刊信息

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex