Single molecule DNA mapping for DNA and CNV analysis

描述（由申请人提供）：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，而不执行全基因组组装。当尝试全基因组组装时，其通过从具有不同插入物大小的许多测序文库产生配对末端测序读段来完成。双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，并提供了有限的信息，二倍体人类基因组的单倍型。类似地，目前基于基于阵列的比较基因组杂交的结构变异扫描不能确定重复区域的基因组位置或鉴定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，自动化的方法，光学映射的一般用途。我们的基因组作图策略始于双链基因组DNA的序列特异性切口，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，或者用设计用于互补单链DNA瓣上发现的特定序列的荧光探针标记。然后将大的（100 kbp至300 kbp）标记的DNA片段在纳米通道中线性化，用于在与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，可以创建针对所问问题的基因组图谱，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段序列，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，人类基因组中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。在这个提议中，我们的目标是开发和优化一个单分子作图方法，使从头序列组装和结构变异分析成为可能。

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.