Single molecule DNA mapping for DNA and CNV analysis

用于 DNA 和 CNV 分析的单分子 DNA 作图

• 批准号: 8292200
• 负责人: Pui-Yan KWOK
• 金额: $ 58万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2013-12-16
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8292200
• 关键词: Bacterial Artificial Chromosomes; Complement; DNA; DNA mapping; DNA-Directed DNA Polymerase; Data; Detection; Development; Diploidy; Disease; Equilibrium; Equipment; Fluorescent Probes; Genome; Genome Mappings; Genomics; Haplotypes; Housing; Human Chromosomes; Human Genome; Image Analysis; Individual; Label; Libraries; Location; Maps; Medical; Methods; Optical Methods; Optics; Primer Extension; Reaction; Reading; Resolution; Scanning; Scheme; Screening procedure; Shotgun Sequencing; Single-Stranded DNA; Site; Solutions; Structure; Surgical Flaps; System; Variant; base; comparative genomic hybridization; design; flexibility; genome sequencing; genome wide association study; nano; nanochannel; public health relevance; scaffold; single molecule

DESCRIPTION (provided by applicant): Two of the major challenges in genome analysis are de novo genome sequence assembly based on "short read" shotgun sequencing and structural variation analysis. At present, most medical sequencing projects and whole genome sequencing projects map the sequencing data onto the reference human genome sequence without performing whole genome assemblies. When whole genome assembly is attempted, it is done by generating paired-end sequencing reads from a number of sequencing libraries with different insert sizes. The paired-end sequences provide the "scaffold" that helps with sequence assembly. However, it increases the complexity of the sequencing project and provides limited information on the haplotypes of the diploid human genome. Similarly, current structural variation scanning based on array-based comparative genomic hybridization is unable to determine the genomic locations of duplicated regions or identify genomic inversions or balanced translocations. We propose to optimize a new, highly flexible, automated method for optical mapping for general use. Our genome mapping strategy starts with sequence specific nicking of double-stranded genomic DNA followed by displacing a short strand of DNA downstream of the nicking site with DNA polymerase. These nicked-flap structures can be labeled with fluorescent dNTPs by a primer extension reaction or with fluorescent probes designed to complement specific sequences found on the single-stranded DNA flaps. The large (100 kbp to 300 kbp) labeled DNA fragments are then linearized in nano-channels for high-throughput, automated imaging and analysis on a system assembled with off-the-shelf equipment. By intelligent probe design, one can therefore create genome maps tailored to the questions being asked, be it local structural variation screening, global structural variation detection, or scaffolding for de novo genome sequence assembly. PUBLIC HEALTH RELEVANCE: As sequencing platforms are producing short-read sequences at extremely high rates, the main obstacle to whole genome sequencing is the inability to assemble the sequencing data accurately and efficiently. Furthermore, structural variations in the human genome are found to be associated with a number of important diseases but genome-wide scanning for these variations is not yet feasible. In this proposal, we aim to develop and optimize a single molecule mapping approach that will make de novo sequence assembly and structural variation analysis possible.

描述（由申请人提供）：基因组分析的两个主要挑战是基于“短读”鸟枪测序和结构变异分析的从头基因组序列组装。目前，大多数医学测序项目和全基因组测序项目将测序数据映射到参考人类基因组序列上，而不进行全基因组组装。当尝试全基因组组装时，是通过从具有不同插入大小的多个测序文库生成双端测序读数来完成的。双末端序列提供了有助于序列组装的“支架”。然而，它增加了测序项目的复杂性，并提供了有关二倍体人类基因组单倍型的有限信息。类似地，当前基于阵列的比较基因组杂交的结构变异扫描无法确定重复区域的基因组位置或识别基因组倒位或平衡易位。我们建议优化一种新的、高度灵活的、自动化的通用光学测绘方法。我们的基因组作图策略首先对双链基因组 DNA 进行序列特异性切口，然后用 DNA 聚合酶置换切口位点下游的短链 DNA。这些切口瓣结构可以通过引物延伸反应用荧光 dNTP 标记，或者用设计用于补充单链 DNA 瓣上特定序列的荧光探针进行标记。然后，大的（100 kbp 至 300 kbp）标记 DNA 片段在纳米通道中线性化，以便在用现成设备组装的系统上进行高通量、自动成像和分析。通过智能探针设计，人们可以根据所提出的问题创建基因组图谱，无论是局部结构变异筛选、全局结构变异检测还是从头基因组序列组装的支架。 公共卫生相关性：由于测序平台以极高的速率产生短读序列，全基因组测序的主要障碍是无法准确有效地组装测序数据。此外，人类基因组的结构变异被发现与许多重要疾病有关，但对这些变异进行全基因组扫描尚不可行。在本提案中，我们的目标是开发和优化单分子作图方法，使从头序列组装和结构变异分析成为可能。