Nanomapping-Assisted Analysis of Human Telomere Regions

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作图技术（“纳米作图”），用于（a）使用基于BAC的方法对复杂的人类亚端粒区域进行有效的图谱辅助下一代测序和组装，以及（B）试验一种用于对来自任何人类基因组的亚端粒DNA进行选择、纳米作图和测序的新方法。通过开发新的nanomapping方法，旨在允许在亚端粒BAC中克隆的数百kb DNA上绘制1 kb分辨率的特征图谱，我们将实现并展示有效的完整下一代测序和亚端粒单倍型组装，包括先前绘制的大型结构变体。将纳米图谱辅助的下一代测序应用于从基因组DNA捕获的大亚端粒DNA片段，将这种方法扩展到直接分析基因组的亚端粒区域，而不需要大插入克隆文库的中间要求。这项多研究者应用整合了Xiao博士及其同事开创的新型DNA片段标记和纳米通道阵列芯片技术，以及Riethman实验室对当前人类亚端粒参考序列组成和序列组织的详细了解。两者都需要测试这种新技术的能力，以大大提高目前的人类参考序列在亚端粒区域，并开发新的方法来理解端粒长度调控和端粒稳定性的顺式控制。Co-PI各自的专业知识完美吻合，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提高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，并为从未克隆的基因组DNA捕获端粒DNA片段和表征打开大门，以允许基于人群的研究这些序列在端粒功能中的作用。更广泛地说，这些实验将为该方法的广泛应用提供原理证明，该方法将高通量高分辨率DNA特征作图与下一代测序方法相结合，以改善参考基因组质量并分析结构变异。

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.