Targeted nanopore sequencing with Cas9-guided adapter ligation

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Current sequencing methods face limitations in achieving high depth and detecting diverse variants.
• Existing assays often struggle with native modification preservation, read length, input DNA requirements, yield, or protocol duration.

Purpose of the Study:

• To introduce nanopore Cas9-targeted sequencing (nCATS) as an advanced enrichment strategy.
• To demonstrate nCATS' capability for simultaneous haplotype-resolved variant detection and methylation analysis.

Main Methods:

• nCATS utilizes targeted DNA cleavage by Cas9 for adapter ligation in nanopore sequencing.
• The method was applied to various samples including cell lines, xenografts, and primary human breast tissues.

Main Results:

• nCATS achieved high median sequencing coverage (675× on MinION, 34× on Flongle).
• The technique requires minimal genomic DNA input (~3 μg) and can target numerous loci concurrently.
• Simultaneous assessment of single-nucleotide variants, structural variations, and CpG methylation was demonstrated.

Conclusions:

• nCATS provides a powerful tool for high-depth, comprehensive variant detection and epigenetic analysis.
• This method enhances the utility of long-read sequencing for both research and clinical applications.