HIGH RESOLUTION ALLELE SPECIFIC EXPRESSION ASSAYS

描述(申请人提供)：基因表达的遗传学研究为基因调控和基因调控网络的架构提供了重要的洞察力，并为识别不同特征背后的分子机制提供了新的机会。目前，这个实验系统使我们能够识别调控变异，并对人类变异的功能谱进行建模。此外，这样的研究对于目前深入了解遗传效应的特定组织背景是基本的。基因类型-组织表达(GTEx)项目现在提出了相当大的未来努力，以表征基因表达的遗传效应的组织特异性；到2016年，GTEx计划收集近900个人的20,000个组织转录本。最大限度地增加这项投资和开发黄金标准资源的关键是应用实验方法来验证发现，特别是表达数量性状基因座(EQTL)。验证eQTL的一种这样的方法是等位基因特异性表达(ASE)测试；与驱动eQTL发现的基因关联不同，等位基因测试暗示个体内存在顺式连锁的调节变体，并且独立于因果变体的群体频率。然而，来自RNA-Seq的ASE数据的一个局限性是，由于基因表达水平的变化，采样深度的差异可能会对它们产生偏差；这一挑战在多组织研究中加剧。为了解决这个问题，我们开发并改进了一种结合基于微流控的多重PCR和深度测序(MmPCR-Seq)的成熟方法来提供等位基因特异性表达(ASE)的深度和高通量分辨率的方法。这种方法为获得ASE数据提供了一种高通量、高效和低成本的解决方案(每个样本&lt；9分钟和&lt；$50)，我们已经证明MmPCR-Seq对低数量(10 Ng)和低质量(RIN=2.8)的RNA有效。在这个项目中，我们的目标是将这种方法应用于验证在96个人的至少20-30个组织中的800个组织特定和共享的eQTL基因。此外，我们验证了ASE模式在50个个体的至少30个组织中罕见的有害和功能丧失的变异。这些数据将受到质量控制，并定期向GTEx统计分析工作组和更大的GTEx联盟发布。我们希望这些数据将有助于eQTL的发现和验证，特别是在低表达或中等表达的组织中，以及在某些组织中可能表现出较弱影响的共享eQTL。此外，这些数据将使有害和LOF变异的敏感组织解释成为可能，目的是提高我们理解系统模式的能力，这些模式可能是变量外显率和疾病表现的基础，以及对来自个人外显体和基因组的可能致病变异进行分类的新方法。由于对遗传变异的组织背景的研究已经并主要由基因表达的遗传研究驱动，因此应用MmPCR-Seq来验证eQTL和ASE的模式是开发高质量调控变异图谱的关键一步。

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.