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6千兆酶的人类基因组进行测序。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)光学作图来创建关于酶的有序限制图，(2)将一组核苷酸探针（LNA探针）与单基因组dna在表面进行杂交，以及(3)算法来解决整个基因组的ppsh（位置杂交测序）问题的“本地化版本”。项目计划分为两个阶段：(1)科学合理性评估中试研究[R21]和(2)大规模系统工程[R33]。R21阶段的目的是首先证明LNA探针杂交位点全基因组定位的合理性，然后证明将这些定位组合成单倍型序列的算法可行性。这两个目标的成功可能性可以从我们的初步工作中推断：(1)假假单倍型和人类4号染色体片段的单倍型定位；(2) DNA荧光成像及其AFM技术验证；(3)我国研究人员在光学制图方面的现有工作。R33阶段旨在通过连续构建(1)高通量光学系统，(2)恶性疟原虫基因组100bp片段测序（小尺寸）初步验证，(3)更复杂的智人基因组100bp片段测序（大尺寸）验证，(4)最终系统工程和整个智人基因组测序验证。

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.