varCUT&Tag: A Method for Simultaneous Identification and Characterization of Sequence Variants in Regulatory Elements and Genes

项目摘要 用于鉴定序列变体的大多数方法集中于使用全基因组覆盖的宽基因组覆盖。 基因组测序（WGS），通常在~50倍的深度，或更高深度的编码区测序（ 外显子组）。然而，典型的WGS策略忽略了 大多数罕见（< 5%）的序列变异，而WGS在识别罕见变异所需的深度可能是成本- 禁止的。外显子组测序方法可以以合理的成本提供高覆盖率的编码区 但是错过了发生在编码区之外的绝大多数突变。除了编码区，基因 顺式调节区（克雷斯）的变异可以通过改变细胞和组织的功能而对细胞和组织的功能产生重大影响。 关键基因的表达。还没有用于高深度CRE测序的通用富集策略。 因为基因组的不同区域在不同的细胞类型中充当克雷斯，需要不同的 在每个细胞谱系中富集的序列。利用克雷斯由同一组标记的事实， 不同细胞类型的表观遗传标记，我们将利用我们创建的最新表观遗传分析技术， 开发一种称为varCUT&Tag的新方法，用于高覆盖率测序和鉴定罕见变异体 在克雷斯和基因体中（UG 3期）。我们的方法的一个关键特征将是同时 识别每个变体周围的表观遗传标记，从而能够对效应进行功能表征 体细胞突变对CRE功能的影响我们还将开发这种方法的单细胞版本， 包括单细胞RNA测序组件，允许序列变体对基因的影响， 表情要识别。因此，我们不仅能够在不同的细胞中鉴定罕见的CRE变体， 组织类型，但我们将同时优先考虑改变CRE功能的变体，以供未来研究。使用此 方法，我们将在SMaHT网络中工作，以识别和表征来自众多CRE变体的CRE变体。 人类组织，揭示变异对组织异质性和细胞谱系的影响（使用scVarCUT&Tag）， 并参与网络内的合作研究，以增加变异发现（所有UH 3阶段）。总之， varCUT&Tag将填补我们对体细胞序列变异的理解中的一个主要空白， 深入了解它们在不同人体组织中的作用。

PROJECT SUMMARY Most methods for identification of sequence variants focus either on broad genomic coverage using whole genome sequencing (WGS), typically at depths of ~50-fold, or higher depth sequencing of coding regions (the exome) after enrichment using oligonucleotide hybridization strategies. However, typical WGS strategies miss most rare (< 5%) sequence variants, while WGS at depths required to identify rare variants can be cost- prohibitive. Exome sequencing approaches can provide high coverage of coding regions at reasonable cost but miss the vast majority of mutations that occur outside of coding regions. Besides coding regions, genetic variation in cis regulatory regions (CREs) can have a major impact on cell and tissue function by altering expression of critical genes. No general enrichment strategies for high depth CRE sequencing have been described because different regions of the genome act as CREs in different cell types, necessitating different sequences to be enriched in each cell lineage. Drawing on the fact that CREs are marked by the same set of epigenetic marks in different cell types, we will leverage recent epigenetic profiling technologies we created to develop a new method called varCUT&Tag for high coverage sequencing and identification of rare variants within CREs and gene bodies (UG3 phase). A critical feature of our approach will be the simultaneous identification of epigenetic marks surrounding each variant, enabling functional characterization of the effects of somatic mutations on CRE function. We will also develop a single cell version of this approach, which includes a single cell RNA sequencing component, allowing the effects of sequence variants on gene expression to be identified. As a result, we will be able to not only identify rare CRE variants in diverse cell and tissue types, but we will simultaneously prioritize variants that alter CRE function for future studies. Using this approach, we will work within the SMaHT network to identify and characterize CRE variants from numerous human tissues, uncover the effects of variants on tissue heterogeneity and cell lineage (using scVarCUT&Tag), and engage in collaborative studies within the Network to increase variant discovery (all UH3 phase). In sum, varCUT&Tag will fill a major gap in our understanding of somatic sequence variants and provide mechanistic insight into their effects in diverse human tissues.