Tagmentation-based Indexing for Methylation Sequencing as a novel method of high-throughput methylation clock measurement

基于标签的甲基化测序索引作为高通量甲基化时钟测量的新方法

基本信息

批准号：
10273233
负责人：
DAVID A. SINCLAIR
金额：
$ 62.63万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-17 至 2023-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10273233
关键词：
Address Age Aging Animals Bar Codes Biological Biological Aging Biological Assay Biological Markers Biology Biotin Cell physiology Cells Cost Measures DNA DNA Methylation DNA Transposable Elements DNA methylation profiling Development Disease Effectiveness Embryonic Development Epigenetic Process Feasibility Studies Genetic Goals Health Healthcare Human In Vitro Individual Intervention Intervention Studies Libraries Light Longevity Malignant Neoplasms Mammals Measurement Measures Mediating Medical Oncology Medical Research Medicine Methods Methylation Modification Monitor Mus Pharmacology Preparation Process Protocols documentation RNA Repression Reproducibility Research Role Sampling Signal Transduction Site Speed Technology Ticks Tissues Variant X Inactivation animal tissue anti aging base bisulfite bisulfite sequencing cancer diagnosis carcinogenesis cell type combinatorial cost cost effective design epigenetic marker epigenomics experimental study genetic association healthspan high throughput screening human tissue imprint indexing insight mortality novel personalized medicine single cell analysis single cell sequencing tool tumor

项目摘要

PROJECT SUMMARY Despite the importance of the epigenetic clock in epigenomics research and to human health, methods to measure methylation age remain for the most part expensive and not amenable to high throughput assays. We are developing a new method, called Tagmentation-based Indexing for MEthylation sequencing (TIME-seq), for cost-effective high throughput methylation sequencing of epigenetic clock sites. TIME-seq will reduce costs by attaching barcodes and fragmenting DNA in a single step (tagmentation) and by enriching for clock sites using biotinylated RNA baits. Feasibility studies demonstrate that TIME-seq is compatible with multiplexing and successfully enriches for clock sites. Our first goal is to optimize TIME-seq as a high-throughput assay for measuring DNA methylation-based biomarkers. We will accomplish this by optimizing the design of biotin-RNA baits to accommodate diverse DNA methylation clocks from humans and mice. We will also compare the reproducibility and accuracy of TIME-seq to those of established assays for epigenetic clock analysis. We predict that TIME-seq will allow for an increase in the scale of methylation sequencing experiments by at least two orders of magnitude and make clock measurement more accessible by lowering the cost per sample. Moreover, we will adapt TIME-seq for use on single cells. Currently, DNA methylation clocks measure the average methylation age of many cells in a tissue, despite the possibility that only a small number of cell types within a tissue contribute to the majority of the signal. Therefore, we will develop a single-cell TIME-seq (scTIME-seq) protocol based on combinatorial indexing to understand epigenetic aging at the cellular level. This method will allow for measurement of cell-to-cell variation at methylation clock sites and will provide insight into the mechanisms of aging. Ultimately, we aim to increase access to targeted methylation sequencing, and envision it becoming a standard tool used in research and personalized medicine.

项目概要尽管表观遗传时钟在表观基因组学研究和人类健康中很重要，但测量甲基化年龄在很大程度上仍然昂贵且不适合高通量测定。我们正在开发一种新方法，称为基于标记的甲基化测序索引 (TIME-seq)，表观遗传时钟位点的经济高效的高通量甲基化测序。 TIME-seq 将降低成本通过一步（标签化）附加条形码和片段化 DNA，并使用以下方法富集时钟位点生物素化的RNA诱饵。可行性研究表明 TIME-seq 与多路复用兼容成功丰富了时钟站点。我们的首要目标是优化 TIME-seq 作为高通量检测测量基于 DNA 甲基化的生物标志物。我们将通过优化生物素-RNA 的设计来实现这一目标适应人类和小鼠不同 DNA 甲基化时钟的诱饵。我们还将比较 TIME-seq 与已建立的表观遗传时钟分析方法的再现性和准确性。我们预测 TIME-seq 将允许甲基化测序实验的规模增加至少两个数量级幅度并通过降低每个样本的成本使时钟测量更容易实现。此外，我们将调整 TIME-seq 以用于单细胞。目前，DNA 甲基化时钟测量平均甲基化组织中许多细胞的年龄，尽管组织中可能只有少数细胞类型贡献了大部分信号。因此，我们将开发单细胞TIME-seq（scTIME-seq）协议基于组合索引来了解细胞水平的表观遗传衰老。该方法将允许测量甲基化时钟位点的细胞间变异，并将深入了解甲基化时钟位点的机制老化。最终，我们的目标是增加靶向甲基化测序的机会，并设想它成为一个用于研究和个性化医疗的标准工具。