Technology for measuring telomere length of individual chromosomes of single cancer cells


Year of Award:
2019
Status:
Active
Award Type:
R21
Project Number:
CA223727
RFA Number:
RFA-CA-18-002
Technology Track:
Molecular & Cellular Analysis Technologies
PI/Project Leader:
HUANG, XIAOHUA
Other PI or Project Leader:
Not Applicable
Institution:
UNIVERSITY OF CALIFORNIA, SAN DIEGO
PROJECT SUMMARY/ABSTRACT: Telomere lengths and maintenance play very important roles in cancer, aging and other human diseases.The lengths of human telomeres are known to be highly heterogeneous and variable for different cell types/ageand individuals, ranging from 0.5 kb to 20 kb in normal cells, and usually much shorter in most cancer cells. Itis also known that the shortest telomere, not the average telomere length, is critical for cell viability andchromosome stability, and individual telomere length heterogeneity and telomere-driven genomic instabilitymay contribute to early carcinogenesis. However, the most common methods (TRF, qPCR and Q-FISH) fortelomere length measurements can only be used to estimate or infer the average length of the telomeres frommany cells with very limited sensitivity, resolution and accuracy. Even more surprisingly, none is capable ofmeasuring the length of the telomeres of all individual chromosomes and of single cells. The deficiency hasgreatly hammered many telomere-related association studies and the use of telomere lengths as biomarkersfor clinical diagnosis of cancer and for prognosis of cancer treatments. To move the field of telomere biologyforward and to enable the routine clinical use of telomeres as cancer biomarkers, we need a scalabletechnology for low-cost absolute length measurements of the telomeres of individual chromosomes in singlecells. In this project, we propose to develop a technology (TeloMeSeq) for measuring the absolute lengths ofthe individual telomeres of all chromosomes in single human cells with single-nucleotide resolution in ascalable and economic manner. We will leverage on our NEM-SDA (nicking endonuclease mediated strand-displacement amplification) technology that has demonstrated to be capable of linear amplification of verygenomic DNA molecules independent of length to develop a one-tube procedure for the unbiased linearamplification of all telomeres of single human cell and subsequent efficient sequencing library construction. Wewill also leverage the powerful Pacific Biosciences' single-molecule real-time sequencing technology (PacBioSMRT) that offers high throughput sequencing with very long reads (median read lengths > 20 kb) to sequencethe amplified telomeres in full length to identify the individual telomeres and to determine their lengths bycounting the number of telomere AATGGG repeats. We aim to demonstrate a proof of concept of theTeloMeSeq technology using single cells from normal human leukocytes and IMR90 cell line, and cancer celllines (Jurkat and LNCaP). If successfully developed, our technology will enable the absolute lengthmeasurements of all individual telomeres in single human cells with single-base resolution, far superior toexisting methods. Our TeloMeSeq technology has a great potential to transform telomere biology studies andclinical practice where telomeres are used as biomarkers for cancer diagnosis and staging, aging and otherhuman diseases.