Haplotype-specific sequence assembly and breakpoint identification of the 22q11.2 deletion

We have developed a generally applicable method based on CRISPR/Cas9-targeted ultra-long read sequencing (CTLR-Seq) to completely and haplotype-specifically resolve, at base-pair resolution, large, complex, and highly repetitive genomic regions that had been previously impenetrable to next-generation sequencing analysis such as large segmental duplication (SegDup) regions and their associated genome rearrangements that stretch hundreds of kilobases. Our method combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to haplotype-specifically isolate intact large (100-2000 kb) target regions that encompass previously unresolvable genomic sequences. These target fragments are then sequenced (amplification-free) with up to 200x on-target coverage using nanopore sequencing, allowing for the complete assembly of the complex genomic regions of interest at single base-pair resolution. We applied CTLR-Seq to resolve the exact sequence of segmental duplication-mediated rearrangements that constitute the boundary regions of the 22q11.2 deletion CNV. We then perform de novo assembly to resolve, for the first time, at single base-pair resolution, the sequence rearrangements of the 22q11.2 CNVs, and mapped, also for the first time, the exact locations of the breakpoint locations.