Drop-BS: high-throughput single-cell bisulfite sequencing on a microfluidic droplet platform

Drop-BS：微流控液滴平台上的高通量单细胞亚硫酸氢盐测序

基本信息

批准号：
9592639
负责人：
Chang Lu
金额：
$ 19.23万
依托单位：
VIRGINIA POLYTECHNIC INST AND ST UNIV
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-21 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9592639
关键词：
Biological Assay Breast Cancer Patient Cell Line Cell physiology Cells Complex Cytolysis DNA DNA Methylation Data Data Set Development Devices Disease Drops Epigenetic Process Future Genetic Fingerprintings Genome Genomics Gold Human Libraries Mammals Mammary Neoplasms Manuals Microfluidic Microchips Microfluidics Modification Nucleosomes Nucleotides Patients Population Positioning Attribute Preparation Procedures Protocols documentation Reaction Regulation Research Personnel Resolution Running Sampling Technology Testing Time Trees Tube Untranslated RNA Xenograft procedure base bisulfite bisulfite sequencing cost epigenomics forest genome-wide histone modification innovation interest malignant breast neoplasm methylome methylomics molecular subtypes mouse model next generation sequencing tool tumor tumor heterogeneity whole genome

项目摘要

Project Summary Cellular processes are regulated by a complex interplay among different layers of epigenetic information, including DNA methylation, histone modification, nucleosome position, and expression of noncoding RNA. DNA methylation is the best studied epigenetic modification in mammals. The genome-wide DNA methylation profile (i.e., DNA methylome) is established early in development for regulation and perturbed methylome is critically involved in disease development. There have been a number of genome-wide technologies developed for profiling DNA methylomes. Whole genome bisulfite sequencing (WGBS) is generally considered the gold standard for DNA methylation analyses. The approach generates methylomic profile with single-nucleotide resolution. Conventional WGBS requires a large amount of DNA. Although single-cell bisulfite sequencing was demonstrated recently, these assays were based heavily on manipulation in tubes/well plates and manual procedures, making rapid processing of a large number (>1000) of single cells difficult. In this project, we will develop a high-throughput single- cell bisulfite sequencing technology, referred to as Drop-BS, based on a microfluidic droplet platform. We will test and validate the technology by examining intratumoral heterogeneity in single-cell DNA methylomes of breast tumors from patients and patient-derived xenograft (PDX) mouse model.

项目概要细胞过程受到不同表观遗传层之间复杂的相互作用的调节信息，包括 DNA 甲基化、组蛋白修饰、核小体位置和非编码RNA的表达。 DNA甲基化是研究最充分的表观遗传修饰哺乳动物。建立全基因组 DNA 甲基化谱（即 DNA 甲基化组）处于发育早期的调控和受干扰的甲基化组与疾病密切相关发展。已经开发了许多用于分析的全基因组技术 DNA 甲基化组。全基因组亚硫酸氢盐测序（WGBS）通常被认为是 DNA 甲基化分析的金标准。该方法生成甲基组谱单核苷酸分辨率。传统的 WGBS 需要大量 DNA。虽然最近展示了单细胞亚硫酸氢盐测序，这些测定很大程度上基于管/孔板中的操作和手动程序，使得快速处理大量单细胞数量（>1000）困难。在这个项目中，我们将开发一种高通量单基于微流控液滴的细胞亚硫酸氢盐测序技术，简称Drop-BS 平台。我们将通过检查肿瘤内异质性来测试和验证该技术来自患者和患者来源的异种移植物 (PDX) 的乳腺肿瘤的单细胞 DNA 甲基化组鼠标模型。