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Targeted nanopore sequencing with Cas9-guided adaptor ligation

Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods(1-4) are limited by the loss of native modifications, short read length, high input requirements, low yield, or long protocols. H...

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Detalles Bibliográficos
Autores principales: Gilpatrick, Timothy, Lee, Isac, Graham, James E., Raimondeau, Etienne, Bowen, Rebecca, Heron, Andrew, Downs, Bradley, Sukmar, Saraswati, Sedlazeck, Fritz J, Timp, Winston
Formato: Online Artículo Texto
Lenguaje:English
Publicado: 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7145730/
https://www.ncbi.nlm.nih.gov/pubmed/32042167
http://dx.doi.org/10.1038/s41587-020-0407-5
Descripción
Sumario:Despite recent improvements in sequencing methods, there remains a need for assays that provide high sequencing depth and comprehensive variant detection. Current methods(1-4) are limited by the loss of native modifications, short read length, high input requirements, low yield, or long protocols. Here, we describe nanopore Cas9-targeted sequencing (nCATS), an enrichment strategy that uses targeted cleavage of chromosomal DNA with Cas9 to ligate adaptors for nanopore sequencing. We show that nCATS can simultaneously assess haplotype-resolved single-nucleotide variants (SNVs), structural variations (SVs) and CpG methylation. We apply nCATS to four cell lines, a cell-line-derived xenograft, and 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. nCATS requires only ~3μg 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.