Targeted nanopore sequencing with Cas9-guided adapter ligation

Nat Biotechnol. 2020 Apr;38(4):433-438. doi: 10.1038/s41587-020-0407-5. Epub 2020 Feb 10.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / metabolism*
  • Cells, Cultured
  • Chromosomes, Human / genetics
  • Genetic Loci / genetics
  • Genetic Variation
  • Genotype
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Nanopore Sequencing / methods*
  • RNA, Guide, CRISPR-Cas Systems
  • Sequence Analysis, DNA


  • CRISPR-Associated Protein 9