Open AccessTargeted nanopore sequencing with Cas9-guided adapter ligation
Author(s) -
Timothy Gilpatrick,
Isac Lee,
James E. Graham,
Etienne Raimondeau,
Rebecca Bowen,
Andrew J. Heron,
Bradley M. Downs,
Saraswati Sukumar,
Fritz J. Sedlazeck,
Winston Timp
Publication year - 2020
Publication title -
nature biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 15.358
H-Index - 445
eISSN - 1546-1696
pISSN - 1087-0156
DOI - 10.1038/s41587-020-0407-5
Subject(s) - nanopore sequencing , minion , adapter (computing) , dna sequencing , cas9 , deep sequencing , computational biology , dna sequencer , crispr , biology , dna , genome , genetics , computer science , gene , computer hardware
Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods 1-4 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.