Nanomapping-Assisted Analysis of Human Telomere Regions

人类端粒区域的纳米绘图辅助分析

• 批准号: 8654354
• 负责人: Harold RIETHMAN
• 金额: $ 19.46万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-20 至 2015-08-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8654354
• 关键词: Affect; Aging; Alleles; Apoptosis; Base Sequence; Cell Aging; Chromatin; Chromosomes; Chromosomes, Artificial, Yeast; Code; Complex; DNA; DNA copy number; Data; Disease; Elements; Environment; Epigenetic Process; Functional RNA; Functional disorder; Gene Expression; Genome; Genomic DNA; Genomic Instability; Genomics; Haplotypes; Heart Diseases; Human; Human Chromosomes; Human Genome; Individual; Label; Lead; Left; Length; Libraries; Malignant Neoplasms; Maps; Methods; Preparation; Pulsed-Field Gel Electrophoresis; RNA; Regulation; Relative (related person); Research Personnel; Resolution; Resources; Role; Source; Techniques; Technology; Testing; Transcript; Variant; base; design; improved; insertion/deletion mutation; nanochannel; new technology; novel; novel strategies; population based; public health relevance; research study; single molecule; success; telomere

DESCRIPTION (provided by applicant): We propose to modify and extend a new, highly accurate high-throughput single-molecule mapping technique ("nanomapping") for (a) efficient map-assisted next-gen sequencing and assembly of complex human subtelomere regions using BAC-based approaches, and (b) piloting a novel method for selection, nanomapping and sequencing of subtelomeric DNA from any human genome. By developing new nanomapping methods designed to permit 1 kb resolution feature maps across hundreds of kb of DNA cloned in subtelomeric BACs, we will enable and demonstrate the efficient full next-gen sequencing and assembly of subtelomeric haplotypes including large structural variants mapped previously. Applying nanomap-assisted next-gen sequencing to large subtelomeric DNA fragments captured from genomic DNA will extend this approach to direct analysis of subtelomeric regions of genomes without an intermediate requirement for large-insert clone libraries. This multi-investigator application integrates the novel DNA fragment labeling and nanochannel array chip technology pioneered by Dr. Xiao and colleagues with the Riethman lab's detailed understanding of the current human subtelomere reference sequence composition and sequence organization. Both are required to test the ability of this novel technology to substantially improve the current human reference sequence in subtelomere regions and to develop novel approaches to understanding cis-control of telomere length regulation and telomere stability. The respective expertise of the Co-PIs dovetail perfectly and create an ideal environment for developing this technology. Successful application of these methods will dramatically improve the quality and alternative allele depth of subtelomeric regions in the current human reference sequence, and open the door to telomeric DNA fragment capture and characterization from uncloned genomic DNA to permit population-based studies of the role of these sequences in telomere function. More broadly, these experiments will provide proof-of-principle for the wide application of this approach combining high-throughput high-resolution DNA feature mapping with next- gen sequencing methods to improve reference genome quality and analyze structural variation.

描述（由申请人提供）： 我们建议修改和扩展一种新的，高度准确的高通量单分子映射技术（“纳米绘制”），以使用基于BAC的基于BAC的方法，以及（b）促进选择，纳米绘制和序列的人类基因dna的新颖方法，并使用基于BAC的基于BAC的方法进行复杂的人类亚电位区域的复杂人类亚电位区域的有效绘制的下一代测序和组装。通过开发旨在允许在亚电体BAC中克隆数百个KB的DNA的1 KB分辨率图的新纳米化方法，我们将启用并展示有效的完整的下一代测序和亚电体单倍型，包括先前映射的大型结构变体。将纳米AMAP辅助的下一代测序应用于从基因组DNA捕获的大型亚端粒DNA片段，将将这种方法扩展到对基因组的亚电位区域的直接分析，而无需大型插入克隆文库的中间要求。该多侵袭器应用程序集成了新的DNA片段标记和纳米渠道阵列芯片技术，由Xiao博士和同事与Riethman Lab对当前人类亚端子参考序列组成和序列组织的详细理解相结合。两者都必须测试这项新技术实质上改善亚电位区域中当前人类参考序列的能力，并开发出新的方法来理解端粒长度调节和端粒稳定性的顺式对照。 Co-Pis的各自专业知识完美地吻合，并为开发这项技术创造了理想的环境。这些方法的成功应用将显着改善当前人类参考序列中亚端粒区域的质量和替代等位基因深度，并为端粒DNA片段片段捕获和表征从未悬而未决的基因组DNA允许基于人群的基于人群的研究，以允许这些序列在端粒功能中的作用的研究。更广泛地说，这些实验将为这种方法的广泛应用提供原则证明，该方法将高通量高分辨率的DNA特征映射与下一代测序方法相结合，以提高参考基因组质量并分析结构变化。