Single molecule DNA mapping for genome and CNV analysis

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全基因组结构变异分析。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始于序列特异性标记双链基因组DNA片段与荧光团。荧光标记的大（100 kbp至1 Mbp） DNA片段然后在纳米通道阵列中线性化，以便在市售仪器上进行高通量自动成像和分析。随着越来越多的团队进行大规模的基因组测序和寻找结构变异，一种普通实验室内部可以使用的方法将促进医学基因组学的研究。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。在本提案中，我们的目标是改进和扩展该方法，以轻松地从1000基因组计划中生成1,300个个体，以提供全基因组结构变异数据和这些全基因组测序对象的完整测序数据。

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 genome-wide 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 labeling double-stranded genomic DNA fragments with fluorophores. The fluorescently labeled, large (100 kbp to 1 Mbp) DNA fragments are then linearized in nanochannel arrays for high-throughput, automated imaging and analysis on a commercially available instrument. As more and more groups are performing large-scale genomic sequencing and searching for structural variation, a method that average labs can use in-house will facilitate medical genomics studies. 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. In this proposal, we aim to improve and scale the method to generate, with ease, >300 individuals from the 1000 Genomes Project to provide both genome-wide structural variation data and fully assembled sequencing data on these whole-genome sequenced subjects.