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”霰弹枪测序的从头基因组序列组装和结构变异分析。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，它是通过从许多具有不同插入大小的测序文库中生成成对端测序reads来完成的。成对末端序列提供了“支架”，有助于序列组装。然而，它增加了测序项目的复杂性，并提供了有限的信息，单倍型的二倍体人类基因组。同样，目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，也无法识别基因组倒置或平衡易位。我们建议优化一种新的、高度灵活的、自动化的光学映射方法，以供一般使用。我们的基因组定位策略从双链基因组DNA的序列特异性缺口开始，然后用DNA聚合酶取代缺口位点下游的短链DNA。这些缺口皮瓣结构可以通过引物延伸反应用荧光dNTPs标记，或者用荧光探针设计来补充单链DNA皮瓣上发现的特定序列。大的（100 kbp到300 kbp）标记的DNA片段然后在纳米通道中线性化，用于高通量、自动化成像和分析，系统由现成的设备组装。因此，通过智能探针设计，人们可以根据所要问的问题创建定制的基因组图谱，无论是局部结构变异筛选，全局结构变异检测，还是从头开始的基因组序列组装脚手架。