TY - JOUR
T1 - Targeted nanopore sequencing with Cas9-guided adapter ligation
AU - Gilpatrick, Timothy
AU - Lee, Isac
AU - Graham, James E.
AU - Raimondeau, Etienne
AU - Bowen, Rebecca
AU - Heron, Andrew
AU - Downs, Bradley
AU - Sukumar, Saraswati
AU - Sedlazeck, Fritz J.
AU - Timp, Winston
N1 - Funding Information:
This work was supported by funding from the National Institutes for Health (grant no. R01 HG009190) (National Human Genome Research Institute).
Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature America, Inc.
PY - 2020/4/1
Y1 - 2020/4/1
N2 - Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1–4 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.
AB - Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods1–4 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.
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U2 - 10.1038/s41587-020-0407-5
DO - 10.1038/s41587-020-0407-5
M3 - Article
C2 - 32042167
AN - SCOPUS:85079447140
SN - 1087-0156
VL - 38
SP - 433
EP - 438
JO - Nature biotechnology
JF - Nature biotechnology
IS - 4
ER -