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.

中文翻译：

---

使用CRISPR靶向超长读测序（CTLR-Seq）进行节段复制介导的基因组重排的完整和单元型特异性序列组装

我们已开发出一种基于CRISPR / Cas9靶向的超长读测序（CTLR-Seq）的普遍适用的方法，以碱基对的分辨率完全，单倍型特异地解析原本已经存在的大，复杂和高度重复的基因组区域以前是下一代测序分析所无法理解的，例如大片段重复（SegDup）区域及其相关基因组重排，可延伸数百千碱基。我们的方法结合了体外Cas9介导的基因组切割和脉冲场凝胶电泳，以单倍型特异性分离了完整的大靶标区域（200-550 kb），其中包含以前无法解析的基因组序列。然后使用牛津纳米孔技术对这些目标片段进行测序（无扩增）以产生高达40倍的目标覆盖率的超长读取，允许以单个碱基对的分辨率完整组装感兴趣的复杂基因组区域。我们应用CTLR-Seq解析SegDup重排的确切序列，该序列构成22q11.2缺失CNV和16p11.2缺失与复制CNV的边界区域。这些CNV是已知的最强烈的精神分裂症和自闭症危险因素。然后，我们执行从头组装，首次以单一碱基对的分辨率解析22q11.2和16p11.2 CNV的序列重排，准确绘制出受该基因影响的基因和非编码区CNV适用于不同的运营商。我们应用CTLR-Seq解析SegDup重排的确切序列，该序列构成22q11.2缺失CNV和16p11.2缺失与复制CNV的边界区域。这些CNV是已知的最强烈的精神分裂症和自闭症危险因素。然后，我们执行从头组装，首次以单碱基对分辨率解析22q11.2和16p11.2 CNV的序列重排，准确绘制出受该基因影响的基因和非编码区CNV适用于不同的运营商。我们应用CTLR-Seq解析SegDup重排的确切序列，该序列构成22q11.2缺失CNV和16p11.2缺失与复制CNV的边界区域。这些CNV是已知的最强烈的精神分裂症和自闭症危险因素。然后，我们执行从头组装，首次以单碱基对分辨率解析22q11.2和16p11.2 CNV的序列重排，准确绘制出受该基因影响的基因和非编码区CNV适用于不同的运营商。