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.
Telomeres form the ends of chromosomes;their loss and dysfunction can lead to cellular senescence and apoptosis, impacting organismal aging as well as many diseases including cancer and heart disease. Subtelomeric DNA is among the most variable and complex regions in the human genome, and helps regulate telomere loss and dysfunction;the technology we propose to develop will be uniquely valuable for both efficiently analyzing subtelomeric DNA and helping to understand its role in regulating telomeres.
|McCaffrey, Jennifer; Young, Eleanor; Lassahn, Katy et al. (2017) High-throughput single-molecule telomere characterization. Genome Res 27:1904-1915|
|Young, Eleanor; Pastor, Steven; Rajagopalan, Ramakrishnan et al. (2017) High-throughput single-molecule mapping links subtelomeric variants and long-range haplotypes with specific telomeres. Nucleic Acids Res 45:e73|
|Nelson, David M; Jaber-Hijazi, Farah; Cole, John J et al. (2016) Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability. Genome Biol 17:158|
|Tutton, Stephen; Azzam, Greggory A; Stong, Nicholas et al. (2016) Subtelomeric p53 binding prevents accumulation of DNA damage at human telomeres. EMBO J 35:193-207|
|McCaffrey, Jennifer; Sibert, Justin; Zhang, Bin et al. (2016) CRISPR-CAS9 D10A nickase target-specific fluorescent labeling of double strand DNA for whole genome mapping and structural variation analysis. Nucleic Acids Res 44:e11|
|Sedic, Maja; Skibinski, Adam; Brown, Nelson et al. (2015) Haploinsufficiency for BRCA1 leads to cell-type-specific genomic instability and premature senescence. Nat Commun 6:7505|
|Stong, Nicholas; Deng, Zhong; Gupta, Ravi et al. (2014) Subtelomeric CTCF and cohesin binding site organization using improved subtelomere assemblies and a novel annotation pipeline. Genome Res 24:1039-50|
|Porro, Antonio; Feuerhahn, Sascha; Delafontaine, Julien et al. (2014) Functional characterization of the TERRA transcriptome at damaged telomeres. Nat Commun 5:5379|