Illumina HiSeq 2500 Sequencing System

描述(由申请人提供)： 拉霍亚过敏和免疫学研究所(LIAI)的任务是研究健康和疾病中的免疫细胞。他们感兴趣的领域涉及非常广泛的项目，包括：(I)分析哮喘、胚胎干细胞和癌症中等位基因特有的染色质状态以及DNA甲基化和羟甲基化状态；(Ii)细胞溶解T淋巴细胞分化过程中染色体结构的变化；(Iii)确定从以下来源获得的各种免疫细胞的等位基因特有的全球转录输出 患有哮喘、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，以及人类疾病的相关动物模型。下一代测序是理解这些过程的最新和最强大的工具之一。基因组、表观基因组和转录组的研究提供了关于基因如何对外界环境信号作出反应以及在染色质背景下如何发挥作用和相互作用以维持正常生理或导致疾病的公正信息。由于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，预计全国对这项技术的兴趣激增，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。虽然我们有一个坚实的5500xL测序仪，但它已经被用于容量，而且不适合我们的NIH项目所需的某些应用，包括基因组范围的DNA甲基化和亚硫酸氢法测序分析的羟甲基化；将疾病相关的单核苷酸多态与染色质结构或顺式基因表达的变化联系起来；绘制全基因组的核小体定位图；通过高通量染色体捕获(Hi-C)分析长距离DNA环路；以及从极少数已分类的免疫细胞群中获得高质量的序列信息。因此，我们申请资金购买Illumina HiSeq 2500，这是一种独特的、高通量、具有成本效益的下一代测序系统，具有高灵敏度的延时积分(TDI)行扫描和四个用于双表面成像的CCD传感器，以便每天并行进行高达55千兆基(GB)的大规模测序。它使用简单的合成测序方法和可逆的终止子化学，这是最理想和多功能的测序配置，是广泛的遗传分析和功能基因组应用的理想选择。如果获奖，这台多功能仪器将向所有科学家开放，网址为： LIAI遵循了内部的下一代测序培训课程，毫无疑问，这将使我们在NIH支持的项目上取得及时和有效的进展。固体测序系统在获得长的测序读数长度和研究DNA甲基化状态方面存在挑战；离子PGM测序仪可以产生更长的读数，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.