CRISPR-based targeted haplotype-resolved assembly of a megabase region [WGBS]. CRISPR-based targeted haplotype-resolved assembly of a megabase region [WGBS]

Constructing high-quality haplotype-resolved genome assemblies has substantially improved the ability to detect and characterize genetic variants. A targeted approach providing readily access to the rich information from haplotype-resolved genome assemblies will be appealing to groups of basic researchers and medical scientists focused on specific genomic regions. Here, using the 4.5 megabase, notoriously difficult-to-assemble major histocompatibility complex (MHC) region as an example, we demonstrated an approach to construct haplotype-resolved assembly of the targeted genomic region with the CRISPR-based enrichment. Compared to the results from haplotype-resolved genome assembly, our targeted approach achieved comparable completeness and accuracy with reduced computing complexity, sequencing cost, as well as the amount of starting materials. Moreover, using the targeted assembled personal MHC haplotypes as the reference both improves the quantification accuracy for sequencing data and enables allele-specific functional genomics analyses of the MHC region. Given its highly efficient use of resources, our approach can greatly facilitate population genetic studies of targeted regions, and may pave a new way to elucidate the molecular mechanisms in disease etiology. Overall design: 10x linked-reads, CCS reads and WGBS for Enriched MHC, RNA-seq sequencing and Illumina Human Methylation 850 BeadChip for NA12878

构建高质量单倍型解析基因组组装（haplotype-resolved genome assemblies），已大幅提升了遗传变异的检测与表征能力。
一种可便捷获取单倍型解析基因组组装所蕴含丰富信息的靶向研究策略，将深受聚焦特定基因组区域的基础研究人员与医学科学家的青睐。
本研究以长度为4.5百万碱基、组装难度极高的主要组织相容性复合体（major histocompatibility complex, MHC）区域为范例，展示了一种基于CRISPR富集技术构建靶向基因组区域单倍型解析组装的方法。
相较于全基因组单倍型解析组装的结果，本研究的靶向策略在组装完整性与准确性上表现相当，同时降低了计算复杂度、测序成本以及起始样本需求量。
此外，将靶向组装得到的个体MHC单倍型作为参考序列，不仅提升了测序数据的定量准确性，还可实现MHC区域的等位基因特异性功能基因组学分析。
鉴于该策略对实验资源的高效利用，本方法可极大推动靶向区域的群体遗传学研究，并为阐明疾病发生的分子机制提供新途径。
实验整体设计：针对富集的MHC区域，采用10x连锁读长测序（10x linked-reads）、CCS读长测序（CCS reads）以及全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequencing, WGBS）；针对NA12878样本，采用RNA测序（RNA sequencing, RNA-seq）与Illumina人类甲基化850K芯片（Illumina Human Methylation 850 BeadChip）进行检测。