Methods for Extracting and Analyzing Highly Complex Regions of the Genome - Applications to the IGH locus

作为适应性免疫系统的三大组成部分之一，抗体是必不可少的安装 成功的疾病反应。尽管对个体生殖系抗体大小的保守估计 库的数量级为1014，对慢性疾病状态的免疫反应表明， 抗体的多样性实际上要大得多。鉴于其组织和行为的复杂性，直接 疾病反应和IGH功能之间的联系一直难以捉摸。到目前为止，只有一小部分 免疫系统产生的抗体与特定的疾病刺激有关。此外，委员会认为， 直到最近，只有一个完整的IGH可变基因组区域组装， 基因座的唯一参考序列直到2013年才完成了第二次组装，这一次是从一个 单倍型这两个程序集之间的对比，新的程序集包含的 100kb的以前未知的序列，几个全新的IGHV基因，和许多结构 变异，清楚地表明，在这个基因座的多样性程度是低估。发展新型 算法和实验方法，利用测序和分子生物学的最新进展， 能够高通量提取和解析IGH基因座和其他高变基因组区域。 我们已经开发了“混合”方法，该方法将联合收割机第三代长读段测序 第二代平台的短读技术。我们的团队和其他人已经证明， 这些方法能够在解决复杂结构变化方面远远优于先前的策略， 组装邻接和单体型定相。我们将在这些方法的基础上研究IGH基因座 使用选择性靶向以及通过现有的全基因组测序（WGS）数据。所有信息学 进行这些分析的工具将作为开放源码软件向社区发布。为了 以合理成本有效靶向这些大基因座，我们已经开发了新的富集策略， 是高度特异性的，不依赖于扩增（因此保持表观遗传修饰）， 区域中的DNA邻接（以使组成单元型的准确重建成为可能）。我们将应用此 这将是第一个应用非克隆为基础的靶向任何 这个大小的基因座。此外，鉴于基因组学社区严重依赖短读 高通量靶向基因分型技术，我们将开发一个分子方案和生物信息学 将新的10X Genomics技术与定制IGH捕获面板配对的方法， 短读测序数据的长范围单倍型。这项提案不仅将改善我们的 了解IGH，可能会导致更好的诊断和治疗，但也将提供一个 研究其他高变和生物医学重要基因区域的框架。

As one of the three major components of the adaptive immune system, antibodies are essential to mounting successful disease responses. Though conservative estimates of the size of an individual's germline antibody repertoire are on the order of 1014, immune responses to chronic disease states suggest that the true space of antibody diversity is, in fact, far larger. Given the complexity of its organization and behavior, direct connections between disease responses and IGH function have been elusive. To date, only a small fraction of the antibodies generated by the immune system have been linked to specific disease stimuli. Furthermore, until recently there was only a single complete assembly of the IGH variable genomic region⁠ which served as the sole reference sequence for the locus. Only in 2013 was a second assembly completed, this time from a single haplotype. The contrast between the two assemblies, with the newer assembly containing more than 100kb of previously uncharacterized sequence, several completely new IGHV genes, and numerous structural variations, makes it clear that the degree of diversity at this locus is under-appreciated. We will develop new algorithms and experimental approaches, utilizing recent advances in sequencing and molecular biology, to enable high-throughput extraction and resolution of the IGH locus and other hypervariable genomic regions. We have developed “hybrid” approaches that combine third-generation long-read sequencing technology with short-reads from second-generation platforms. Our group and others have shown that such approaches are capable of far outperforming previous strategies in resolving complex structural variation, assembly contiguity, and haplotype phasing. We will build on these approaches to examine the IGH locus using selective targeting as well as through existing whole genome sequencing (WGS) data. All informatics tools for performing these analyses will be released as open-source software for the community. In order to target such large loci effectively and at reasonable cost, we have developed new enrichment strategies that are highly specific, do not rely on amplification (and therefore maintain epigenetic modifications), and maintain DNA contiguity in the region (to enable accurate reconstruction of constituent haplotypes). We will apply this methodology on available cell lines; this will be the first application of non-clone-based targeting of any genomic locus of this size. In addition, given the heavy reliance of the genomics community on short-read technologies for high-throughput targeted genotyping, we will develop a molecular protocol and bioinformatics approaches to pair the new 10X Genomics technology with a custom IGH capture panel as means to generate long-range haplotypes from short-read sequencing data. Together, this proposal will not only improve our understanding of IGH, potentially leading to better diagnostics and therapeutics, but will also provide a framework for studying other hypervariable, and biomedically important, gene regions.