Targeted nanopore sequencing with Cas9-guided adapter ligation

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

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)433-438
Number of pages6
JournalNature biotechnology
Volume38
Issue number4
DOIs
StatePublished - Apr 1 2020

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ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology
  • Molecular Medicine
  • Biomedical Engineering

Cite this

Gilpatrick, T., Lee, I., Graham, J. E., Raimondeau, E., Bowen, R., Heron, A., Downs, B., Sukumar, S., Sedlazeck, F. J., & Timp, W. (2020). Targeted nanopore sequencing with Cas9-guided adapter ligation. Nature biotechnology, 38(4), 433-438. https://doi.org/10.1038/s41587-020-0407-5