Haplotype Sequencing via Single Molecule Hybridization

描述(申请人提供)：我们寻求资金，在10年内开发一项技术，对包括这两种单倍型的人类大小约6千兆碱基的基因组进行测序。我们的目标是通过成功地集成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图，(2)将一组寡核苷酸探针(LNA探针)与表面的单个基因组DNA杂交，以及(3)解决整个基因组上的PSBH(位置测序)问题的算法。该项目计划分为两个阶段：(1)评估科学稳健性的试点研究[R21]和(2)大型系统工程[R33]。R21阶段的目的是首先证明LNA探针杂交位点全基因组图谱的可靠性，然后证明将这些图谱组合成单倍型序列的算法可行性。这两个目标成功的可能性可以从我们在以下方面的初步工作中推断出来：(1)假丝虫和人类4号染色体片段的单倍型图谱；(2)DNA的荧光成像及其AFM技术的验证；以及(3)我们研究人员在光学图谱方面的现有工作。R33阶段的目的是通过依次构建以下几个阶段来设计最终的系统：(1)高通量光学系统，(2)通过测序恶性疟原虫基因组100bp片段(小片段)进行初步验证，(3)通过对智人基因组100bp片段(大片段)进行测序进行更复杂的验证，(4)最终系统工程和通过对全基因组测序进行验证。

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.