Core B. HighL-Throughput Sequencing Genomics Core

人类基因组计划的完成所带来的惊人进步，新的测序技术和提取基因组DNA（gDNA）的特定片段的新方法，现在已经使得在相对较短的时间内对个体患者中的“外显子组”进行测序成为可能（全外显子组测序，WES）。的核心PI和Co-PI在设计方面非常专业， 实施下一代测序（NGS）实验，并将建立和运行NGS核心B，以生成涉及人类、小鼠和斑马鱼的每个拟议项目的WES数据。 长期以来，人们已经认识到，绝大多数具有强效应的等位基因位于外显子组中，外显子组仅占大多数脊椎动物基因组的1%。捕获和测序人类外显子组的方法是第一个出现的，并且正在改变科学家处理遗传疾病的方式。 捕获和测序小鼠和斑马鱼外显子组的方法将很快推出。在这个核心中，我们将把这些技术应用于结构性脑疾病（SBD）的进化过程中。我们已经成功地生成了WES数据，在> 10倍时产生95%的目标碱基 覆盖对于显性疾病，系统识别杂合变体的能力受到这种覆盖范围的限制，但对于隐性疾病，这一障碍很容易克服。由于人类、小鼠和斑马鱼中纯合突变引起的隐性疾病是本申请的重点，因此我们将非常有能力使用这种方法来鉴定这些物种中的致病突变。 核心B的一个重要方面是不仅与科学项目，而且与核心C（生物信息学核心）和核心A（行政核心）建立密切联系。核心C将开发和利用专门设计用于鉴定纯合潜在有害序列变异的新软件 核心B数据中的PDSV。核心A将支持核心B和核心C的技术基础设施。项目一、二和三将很好地揭示SBD的新机制，并将其转化为关于基本机制的新发现。

The amazing advances brought forward by the completion of the Human Genome Project, new sequencing technologies and new methodologies to extract specific fragments of genomic DNA (gDNA), have now made it possible to sequence the "exome" in an individual patient in a relatively short time-frame (whole exome sequencing, WES). The Core PI and Co-PI of are thoroughly expert in the design and implementation of next-generation sequencing (NGS) experiments, and will establish and operate the NGS Core B to generate WES data across each ofthe proposed Projects, involving human, mouse and zebrafish. It has long been appreciated that the vast majority of alleles with strong effect are located in the exome, which constitutes just 1% of most vertebrate genomes. The methodologies to capture and sequence the exome in humans was the first to emerge and is transforming the way scientists approach genetic disease. Methodologies to capture and sequence the mouse and zebrafish exomes will be available shortly. In this Core, we will bring these technologies to bear on structural brain diseases (SBDs) across the evolutionary spectrum. We have been successful at generating WES data that produces 95% target bases at >10X coverage. For dominant disease, the ability to systematically identify heterozygous variants is limited by this coverage, but for recessive disease, this hurdle is easily overcome. Because recessive disease due to homozygous mutations in humans, mouse and zebrafish is the focus of this application, we will be extraordinarily well-powered to identify causative mutations in these species using this approach. An important aspect of Core B is the close ties that will develop not just to the Scientific Projects but also with Core C (Bioinformatics Core) and Core A (Administrative Core). Core C will develop and utilize new software that is specifically devised for identifying homozygous potentially deleterious sequence variants (PDSVs) in the data from Core B. Core A will support the technological infrastructure of both Core B and Core C. Projects I, II and III will be well-positioned to uncover new mechanisms of SBDs, and translate these into new discoveries about underlying mechanisms.