HIGH RESOLUTION ALLELE SPECIFIC EXPRESSION ASSAYS

高分辨率等位基因特异性表达检测

• 批准号: 9067438
• 负责人: Jin Billy Li
• 金额: $ 30.43万
• 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 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9067438
• 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 Regulation; Genes; Genetic; Genetic Variation; Genetic study; Genome; Genotype; Genotype-Tissue Expression Project; 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; Regulator Genes; Reporting; Resolution; Resources; Sampling; Specificity; Statistical Data Interpretation; 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; transcriptome sequencing; working group

DESCRIPTION (provided by applicant): 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.

描述（由申请人提供）：基因表达的遗传研究为基因调控和基因调节网络的结构提供了重要的见解，以及确定各种特征的分子机制的新机会。目前，这个实验系统使我们能够识别调节变体并建模人类变异的功能谱。此外，此类研究对当前对遗传作用组织特异性背景的见解至关重要。基因型 - 组织表达（GTEX）项目现在提出了将来的巨大努力来表征遗传影响对基因表达的组织特异性。到2016年，GTEX提议从近900个人收集20,000个组织转录组。对于最大化这项投资并开发金标准资源至关重要的是实验方法的应用，这些方法可以验证发现特别表达定量性状基因座（EQTL）。一种验证EQTL的方法是等位基因特异性表达（ASE）的测试；与基因型与驱动EQTL发现的关联不同，等位基因测试暗示了一个个体内的顺式连接调节变体，并且与因果变体的种群频率无关。但是，从RNA-seq中的ASE数据的局限性在于，由于基因表达水平的变化，它们可能会因抽样深度的差异而偏见。多组织研究中加剧的挑战。为了解决这个问题，我们开发了一个测定法，结合了基于微流体的多重PCR和深层测序（MMPCR-SEQ）的既定方法，以提供等位基因特异性表达（ASE）的深层和高通量分辨率。这种方法提供了用于获得ASE数据的高通量，高效且低成本的解决方案（<9分钟，每个样品<$ 50），我们已经证明，MMPCR-Seq在低量（10ng）和低质量（RIN = 2.8）的RNA上有效地工作。在这个项目中，我们旨在将这种方法应用于96个个体中至少20-30个组织中800个组织特异性和共享EQTL基因的验证。此外，我们验证了50个个体中至少30个组织的罕见有害和功能丧失变化的ASE模式。该数据将是质量控制的，并定期发布到GTEX统计分析工作组和较大的GTEX Consortia。我们预计这些数据将有助于发现和验证EQTL，尤其是在低或中间表达的组织中，对于共享EQTL，这可能在某些组织中表现出较弱的效果。此外，该数据将使基于敏感的组织对有害和LOF变异的解释，目的是提高我们理解系统性模式的能力，这些模式可能是可变的渗透性和疾病表现力的基础，以及针对分类的新颖方法，大概是从个人exomes和exomes和基因组中产生的病原性变异。由于对遗传变异的组织环境的研究已经并且主要是由基因表达的遗传研究驱动的，因此MMPCR-SEQ在eqtl验证中的应用和ASE模式是发展调节性变异高质量图的重要步骤。