HIGH RESOLUTION ALLELE SPECIFIC EXPRESSION ASSAYS

高分辨率等位基因特异性表达检测

• 批准号: 8642992
• 负责人: Jin Billy Li
• 金额: $ 71.33万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-22 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8642992
• 关键词: Address; Alleles; Allelic Imbalance; Architecture; Biological Assay; Biology; Cells; Code; Collection; Data; Data Analyses; Data Collection; Disease; Exhibits; Frequencies; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Variation; Genome; Genotype; Goals; Gold; Health; Human; Imagery; Individual; Investigation; Investments; Joints; Link; Maps; Metadata; Methods; Microfluidics; Modeling; Molecular; Nonsense-Mediated Decay; Pattern; Penetrance; Population; Positioning Attribute; Process; Proteins; Quality Control; Quantitative Trait Loci; RNA; RNA Sequences; Regulator Genes; Reporting; Resolution; Resources; Sampling; Solutions; Specificity; Statistical Methods; Statistical Models; System; Testing; Tissues; Triage; Validation; Variant; Work; base; cost; deep sequencing; exome; exome sequencing; genetic regulatory protein; genome sequencing; improved; insight; loss of function; novel strategies; research study; symposium; trait; transcriptome sequencing; working group

Genetic studies of gene expression have provided important insight into the architecture of gene regulation and gene regulatory networks as well as new opportunities to identify molecular mechanisms underlying diverse traits. At the moment, this experimental system enables us to identify regulatory variants and model the functional spectrum of human variation. Furthermore, such studies have been fundamental to current insight into the tissue-specific context of genetic effects. The Genotype-Tissue Expression (GTEx) project is now proposing considerable future effort to characterize the tissue-specificity of genetic effects on gene expression; by 2016, GTEx proposes to collect 20,000 tissue transcriptomes from nearly 900 individuals. Essential to maximizing this investment and developing a gold-standard resource, will be application of experimental methods which can validate discoveries particularly expression quantitative trait loci (eQTL). One such method for validating eQTL are tests of allele-specific expression (ASE); Unlike genotypic association with drives eQTL discovery, allelic tests implicate the presence of a cis-linked regulatory variant within an individual and are independent of the population frequency of a causal variant. However, a limitation of ASE data from RNA-Seq is that they can be biased by differences in sampling depths due to changes in expression level across genes; a challenge that is exacerbated in multi-tissue studies. To address this, we have developed and refined an assay which combines established methods of microfluidics-based multiplex PCR and deep sequencing (mmPCR-Seq) to provide deep and high-throughput resolution of allele-specific expression (ASE). This approach provides a high-throughput, efficient and low-cost solution for obtaining ASE data (<9 minutes and <$50 per sample) and we have demonstrated that mmPCR-Seq works effectively on RNA with low quantity (10ng) and low quality (RIN=2.8). In this project, we aim to apply this approach to the validation of 800 tissue- specific and shared eQTL genes across at least 20-30 tissues in 96 individuals. Furthermore, we validate patterns of ASE for rare deleterious and loss-of-function variation across at least 30 tissues in 50 individuals. This data will be quality-controlled and regularly released to the GTEX Statistical Analysis Working Group and larger GTEx Consortia. We expect that this data will aid discovery and validation of eQTL particularly in tissues with low or intermediate expression and for shared eQTL which may exhibit as weaker effects in some tissues. Furthermore, this data will enable sensitive tissue-based interpretation of deleterious and LoF variation with the goal of improving our ability to understand systemic patterns which may underlie variable penetrance and disease expressivity as well as novel approaches to triage presumably pathogenic variants from personal exomes and genomes. As studies of the tissue context of genetic variation have been and are principally driven by genetic studies of gene expression, application of mmPCR-Seq to the validation of eQTL and patterns of ASE is an essential step towards developing high-quality maps of regulatory variation.

基因表达的遗传学研究为基因调控的结构提供了重要的见解， 基因调控网络以及新的机会，以确定分子机制的基础上， 性状目前，这个实验系统使我们能够识别调节变体并对调节变体进行建模。 人类变异的功能谱。此外，这些研究对当前的洞察力至关重要， 组织特异性的遗传效应。基因型-组织表达（GTEx）项目 建议今后要努力确定基因表达遗传效应的组织特异性; 到2016年，GTEx计划从近900个个体中收集20，000个组织转录组。必须 最大限度地提高这一投资和开发金本位资源，将是实验性的应用， 可以验证发现的方法，特别是表达数量性状基因座（eQTL）。一种这样的方法 用于验证eQTL的是等位基因特异性表达（ASE）测试;与驱动eQTL的基因型关联不同， 发现，等位基因测试暗示在个体内存在顺式连锁的调节变体， 独立于因果变异的群体频率。然而，来自RNA-Seq的ASE数据的局限性 它们可能会因基因间表达水平的变化而导致的采样深度差异而产生偏差; 这一挑战在多组织研究中加剧。为了解决这个问题，我们开发并完善了一个 结合了基于微流体的多重PCR和深度测序的既定方法的测定 （mmPCR-Seq）以提供等位基因特异性表达（ASE）的深度和高通量分辨率。这 方法提供了一种高通量、高效和低成本的解决方案，用于获取ASE数据（<9分钟， <50美元/样品），我们已经证明mmPCR-Seq对低量RNA有效 （10 ng）和低质量（RIN=2.8）。在这个项目中，我们的目标是应用这种方法来验证800个组织- 在96个个体的至少20-30个组织中具有特异性和共享的eQTL基因。此外，我们验证 在50个个体中的至少30个组织中的罕见有害和功能丧失变异的ASE模式。 这些数据将进行质量控制，并定期向GTEX统计分析工作组发布， 更大的GTEx联盟我们希望这些数据将有助于发现和验证eQTL，特别是在组织中 具有低或中等表达以及共享eQTL，其在某些组织中可能表现出较弱的作用。 此外，该数据将能够对有害和LoF变化进行基于组织的敏感解释， 目标是提高我们理解系统模式的能力，这些模式可能是变量的基础， 疾病表现力以及新的方法来分诊可能致病的变异， 外显子组和基因组。由于遗传变异的组织背景研究一直是， 在基因表达的遗传学研究的推动下，mmPCR-Seq在eQTL验证中的应用， ASE模式是开发高质量调控变异图谱的重要一步。