Cas9-Mediated Nanopore Sequencing Enables Precise Characterization of Structural Variants in Genes.

Deletions in the , , and genes are a common cause of familial cerebral cavernous malformations (CCMs). In current molecular genetic laboratories, targeted next-generation sequencing or multiplex ligation-dependent probe amplification are mostly used to identify copy number variants (CNVs). However, both techniques are limited in their ability to specify the breakpoints of CNVs and identify complex structural variants (SVs). To overcome these constraints, we established a targeted Cas9-mediated nanopore sequencing approach for CNV detection with single nucleotide resolution. Using a MinION device, we achieved complete coverage for the genes and determined the exact size of CNVs in positive controls. Long-read sequencing for a and CNV revealed that the adjacent and genes were also partially or completely deleted. In addition, an interchromosomal insertion and an inversion in were reliably re-identified by long-read sequencing. The refinement of CNV breakpoints by long-read sequencing enabled fast and inexpensive PCR-based variant confirmation, which is highly desirable to reduce costs in subsequent family analyses. In conclusion, Cas9-mediated nanopore sequencing is a cost-effective and flexible tool for molecular genetic diagnostics which can be easily adapted to various target regions.