ABSTRACT 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 (200-550 kb) target regions that encompass previously unresolvable genomic sequences. These target fragments are then sequenced (amplification-free) to produce ultra-long reads at up to 40x on-target coverage using Oxford nanopore technology, 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 SegDup rearrangements that constitute the boundary regions of the 22q11.2 deletion CNV and of the 16p11.2 deletion and duplication CNVs. These CNVs are among the strongest known risk factors for schizophrenia and autism. We then perform de novo assembly to resolve, for the first time, at single base-pair resolution, the sequence rearrangements of the 22q11.2 and 16p11.2 CNVs, mapping out exactly the genes and non-coding regions that are affected by the CNV for different carriers.

Complete and haplotype-specific sequence assembly of segmental duplication-mediated genome rearrangements using CRISPR-targeted ultra-long read sequencing (CTLR-Seq)