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Targeted nanopore sequencing with Cas9-guided adapter ligation

Publication

Date: 10th February 2020 | Source: Nature Biotechnology

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

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 675x using a MinION flow cell and 34x using the smaller Flongle flow cell. The nCATS sequencing method 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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