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

项目摘要 尽管表观遗传时钟在表观基因组学研究和人类健康中的重要性， 甲基化年龄的测量在很大程度上仍然是昂贵的，并且不适于高通量测定。我们 正在开发一种新的方法，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成本有效的高通量甲基化测序。TIME-seq将通过以下方式降低成本 在单个步骤（标签片段化）中连接条形码和片段化DNA，并通过使用 生物素化RNA诱饵。可行性研究表明，TIME-seq与多路复用兼容， 成功地丰富了时钟站点。我们的第一个目标是优化TIME-seq作为一种高通量检测， 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 诱饵，以适应不同的DNA甲基化时钟从人类和小鼠。我们还将比较 因此，TIME-seq的重现性和准确性与表观遗传时钟分析的已建立测定法的重现性和准确性相比是非常重要的。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 并通过降低每个样本的成本使时钟测量更容易实现。而且还要 修改TIME-seq以用于单细胞。目前，DNA甲基化时钟测量的是 组织中许多细胞的年龄，尽管组织中只有少量细胞类型 贡献了大部分信号。因此，我们将开发单细胞TIME-seq（scTIME-seq）方案 基于组合索引来理解细胞水平上的表观遗传衰老。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 衰老最终，我们的目标是增加对靶向甲基化测序的访问，并设想它成为一种 用于研究和个性化医疗的标准工具。

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.