Illumina HiSeq 2500 Sequencing System

描述（由申请人提供）： 拉霍亚过敏与免疫学研究所（LIAI）的使命是研究健康和疾病中的免疫细胞。他们感兴趣的领域涵盖了非常广泛的项目，包括：（i）分析哮喘，胚胎干细胞和癌症中的等位基因特异性染色质状态和DNA甲基化和羟甲基化状态;（ii）溶细胞性T淋巴细胞分化过程中染色体结构的变化;（iii）定义从各种免疫细胞获得的等位基因特异性全局转录输出;（iv）分析哮喘，胚胎干细胞和癌症中的DNA甲基化和羟甲基化状态。 患有疾病如哮喘、过敏、镰状细胞病、癌症、心血管疾病、糖尿病和各种自身免疫性疾病的患者，以及来自人类疾病的相关动物模型。下一代测序是了解这些过程的最新和最强大的工具之一。对基因组、表观基因组和转录组的研究提供了关于基因如何响应外部环境信号和在染色质背景下相互作用以维持正常生理或引起疾病的公正信息。预计全国范围内对这项技术的兴趣激增，干扰了NIH资助的LIAI研究人员进入当地甚至国家设施进行下一代测序，迫使他们支付高昂的管理费用，并等待数周才能对他们的样本进行测序。虽然我们有SOLiD 5500 xL测序仪，但这已经被使用到容量，而且不适合我们资助的NIH项目所需的某些应用，包括通过亚硫酸氢盐测序分析的全基因组DNA甲基化和羟甲基化;将疾病相关的单核苷酸多态性与染色质结构或顺式基因表达的变化相关联;绘制全基因组核小体定位图;通过高通量染色体捕获（Hi-C）分析长距离DNA成环;以及从非常少量的分选免疫细胞群体获得高质量的序列信息。因此，我们正在申请购买Illumina HiSeq 2500的资金，这是一种独特的，高通量的，具有高成本效益的下一代测序系统，具有高灵敏度的时间延迟积分（TDI）线扫描和四个CCD传感器，用于双表面成像，每天并行测序高达55千兆字节（Gb）。它使用一种简单的合成测序方法，具有可逆终止子化学，最理想和通用的测序配置，非常适合广泛的遗传分析和功能基因组学应用。如果获奖，这种多功能仪器将提供给所有科学家， LIAI在内部下一代测序培训课程之后，无疑将使我们能够在NIH支持的项目上取得及时有效的进展。SOLiD测序系统在实现长测序读段长度和研究DNA甲基化状态方面存在挑战; Ion PGM测序仪可以产生更长的读段，但吞吐量要低得多。

DESCRIPTION (provided by applicant): Project Summary/Abstract The mission of the La Jolla Institute for Allergy & Immunology (LIAI) is the study of immune cells in health and disease. Their areas of interest cover an extremely wide spectrum of projects including: (i) analysis of allele- specific chromatin state and DNA methylation and hydroxymethylation status in asthma, embryonic stem cells and cancer; (ii) changes in chromosome architecture during the differentiation of cytolytic T lymphocytes; (iii) defining allele-specific global transcriptional output from a variety of immune cells obtained from patients with diseases such as asthma, allergy, sickle cell disease, cancer, cardiovascular disease, diabetes and various autoimmune diseases, as well as from relevant animal models of human disease. Next-generation sequencing is one of the newest and most powerful tools to understand these processes. The study of genomes, epigenomes and transcriptomes provides unbiased information on how genes function and interact with one another in response to outside environmental signals and in the chromatin context to maintain normal physiology or to cause disease. The expected surge of interest in this technology nationwide has inter- fered with the access of NIH-funded LIAI investigators to local and even national facilities for next-generation sequencing, forcing them to pay high overhead costs and wait many weeks for their samples to be sequenced. Although we have a SOLiD 5500xL sequencer, this is already being used to capacity and moreover is not suited for certain applications needed for our funded NIH projects, including genome-wide DNA methylation and hydroxymethylation by bisulfite sequencing analysis; relating disease-associated single-nucleotide poly- morphisms to changes in chromatin structure or gene expression in cis; mapping nucleosome positioning genome-wide; analysis of long-range DNA looping by Hi-throughput chromosome capture (Hi-C); and obtaining high-quality sequence information from very small numbers of sorted immune cell populations. We are therefore requesting funds for purchase of an Illumina HiSeq 2500, a unique, high-throughput, cost effective next-generation sequencing system with highly sensitive Time Delayed Integration (TDI) line scanning and four CCD sensors for dual surface imaging to sequence massively in parallel up to 55 gigabases (Gb) per day. It uses a straightforward sequencing-by-synthesis method with reversible terminator chemistry, the most desirable and versatile sequencing configuration, ideal for a wide range of genetic analyses and functional genomics applications. If awarded, this versatile instrument will be available to all scientists at LIAI following an in-house next-generation sequencing training course, and will undoubtedly allow us to make timely and efficient progress on our NIH-supported projects. The SOLiD sequencing system presents challenges in achieving long sequencing read lengths and studying DNA methylation status; the Ion PGM sequencer can produce longer reads but through-put is much lower.