Title | Targeted nanopore sequencing with Cas9-guided adapter ligation. |
Publication Type | Journal Article |
Year of Publication | 2020 |
Authors | Gilpatrick, T, Lee, I, Graham, JE, Raimondeau, E, Bowen, R, Heron, A, Downs, B, Sukumar, S, Sedlazeck, FJ, Timp, W |
Journal | Nat Biotechnol |
Volume | 38 |
Issue | 4 |
Pagination | 433-438 |
Date Published | 2020 Apr |
ISSN | 1546-1696 |
Keywords | Animals, Cells, Cultured, Chromosomes, Human, CRISPR-Associated Protein 9, Genetic Loci, Genetic Variation, Genotype, High-Throughput Nucleotide Sequencing, Humans, Nanopore Sequencing, RNA, Guide, Kinetoplastida, Sequence Analysis, DNA |
Abstract | Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods 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. |
DOI | 10.1038/s41587-020-0407-5 |
Alternate Journal | Nat Biotechnol |
PubMed ID | 32042167 |
PubMed Central ID | PMC7145730 |
Grant List | T32 CA130840 / CA / NCI NIH HHS / United States T32 CA153952 / CA / NCI NIH HHS / United States R01 HG009190 / HG / NHGRI NIH HHS / United States T32 GM007057 / GM / NIGMS NIH HHS / United States T32 GM136577 / GM / NIGMS NIH HHS / United States |
Targeted nanopore sequencing with Cas9-guided adapter ligation.
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