Single molecule DNA mapping for DNA and CNV analysis

描述(申请人提供)：基因组分析中的两个主要挑战是基于短读鸟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，通过从具有不同插入大小的多个测序文库中产生配对末端测序读数来完成。成对的末端序列提供了帮助序列组装的“支架”。然而，它增加了测序项目的复杂性，并提供了关于二倍体人类基因组单倍型的有限信息。同样，目前基于阵列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、自动化的通用光学测绘方法。我们的基因组作图策略首先是对双链基因组DNA进行序列特异性划痕，然后用DNA聚合酶取代划痕部位下游的一条DNA短链。通过引物延伸反应或设计用于补充单链DNA瓣上的特定序列的荧光探针，可以用荧光dNTPs标记这些缺口瓣结构。然后在纳米通道中线性化标记的DNA大片段(100 KBP到300 KBP)，以便在由现成设备组装的系统上进行高通量、自动化成像和分析。因此，通过智能探针设计，人们可以创建针对所提出的问题量身定做的基因组图，无论是局部结构变异筛选、全球结构变异检测，还是重新组装基因组序列的脚手架。 与公共卫生相关：由于测序平台以极高的速度产生短读序列，全基因组测序的主要障碍是无法准确和有效地收集测序数据。此外，人类基因组中的结构变异被发现与许多重要的疾病有关，但对这些变异进行全基因组扫描还不可行。在这项提案中，我们的目标是开发和优化一种单分子作图方法，使从头开始的序列组装和结构变异分析成为可能。

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.