Haplotype Sequencing via Single Molecule Hybridization

描述（由申请人提供）：我们寻求资金，在10年内开发一种技术，对包括两种单倍型在内的约6千兆碱基的人类大小的基因组进行测序。我们的目标是通过成功地整合三种不同的组件技术来实现这些目标：（1）光学映射以创建关于酶的有序限制性图谱，（2）寡核苷酸探针（LNA探针）池与表面上的单基因组DNA的杂交，以及（3）在整个基因组上解决PSBH（通过杂交的位置测序）问题的“局部版本”的算法。 该项目计划分为两个阶段：（1）评估科学合理性的试点研究[R21]和（2）大规模系统工程[R33]。R21阶段旨在首先证明LNA探针杂交位点的全基因组作图的可靠性，然后证明将这些图谱组合成单倍型序列的算法可行性。这两个目标的成功潜力可以从我们的初步工作中推断出来：（1）T。图1：（2）DNA的荧光成像及其AFM技术的验证;（3）我们的研究人员在光学制图方面的现有工作。R33阶段的目标是通过连续构建：（1）高通量光学系统，（2）通过测序恶性疟原虫基因组（小尺寸）的100 bp片段进行初步验证，（3）通过测序H. sapiens基因组（大尺寸），（4）通过对整个H.智人基因组

DESCRIPTION (provided by applicant): We seek funding to develop, within 10 years, a technology to sequence a human size genome of about 6 Gigabases including both haplotypes. We aim to accomplish these goals by successfully integrating three different component technologies: (1) Optical Mapping to create Ordered Restriction Maps with respect to an enzyme, (2) Hybridization of a pool of oiigonucleotide probes (LNA probes) with Single Genomic DNAs on surface, and (3) Algorithms to solve "localized versions" of PSBH (Positional Sequencing by Hybridization) problems over the whole genome. The project is planned in two stages: (1) Pilot Study to Assess Scientific Soundness [R21] and (2) Large-Scale System Engineering [R33]. The R21 phase aims to demonstrate first the soundness of whole-genome mapping of LNA probe hybridization sites, and then algorithmic feasibility of combining these maps into haplotype sequences. The potential for success of these two aims may be inferred from our preliminary work on (1) haplotype mapping of T. pseudonana and a segment of human chromosome 4; (2) fluorescent imaging of DNA and its validation by AFM technology; and (3) existing body of work on optical mapping by our investigators. The R33 phase aims to engineer the final system by constructing in succession: (1) high throughput optical system, (2) preliminary validation by sequencing 100bp segment of P. falciparum genome (small-size), (3) more complex validation by sequencing 100bp segment of H. sapiens genome (large-size), (4) final system engineering and validation by sequencing the entire H. sapiens genome.