In this application, we seek to develop a transforming technology that will revolutionize epigenomic studies. The approach builds upon a technology we have developed in the past that allowed us to characterize individual DNA molecules in a complex mixture by fluorescence imaging at a rate exceeding 4,000 molecules per minute using femtogram quantities of DNA. These previous fluorescence-imaging studies used phage DNA markers labeled with intercalating dyes for a proof-of-principle test for high throughput single DNA molecule analysis. In three Aims, we propose to extend our existing technology in a stepwise and systematic way, to (a) analyze methylation on DNA and multiple epigenetic marks simultaneously in mammalian chromatin;(b) do this using chromatin isolated from extremely low abundance sources, such as preimplantation embryos and laser microdissected tumors;and (c) sort and recover DNAs carrying specific combinations of marks for subsequent high throughput DNA sequencing. If successful, our efforts will revolutionize epigenomic studies by enabling whole epigenome profiling of single cells;increasing the sensitivity of epigenetic modification detection by several orders of magnitude;facilitating simultaneous monitoring of multiple epigenetic modifications;and providing functional correlates between epigenetic states and gene expression competence. This technology will greatly enhance the capabilities of the Reference Epigenome Mapping Centers. PUBLIC HEALTH RELEVANCE: It has become abundantly clear during the past 20 years that epigenetic alterations to the genome can influence development and health as profoundly as mutagenesis of the genome. One of the most dramatic examples is the fact that methylation of DNA at the promoter of the p16 tumor suppressor is as effective at silencing the gene as mutations to the body of the gene itself and that both events contribute to the development and progression of colorectal cancer [1]. Importantly, unlike mutations, epigenetic silencing of p16 can be reversed pharmacologically, with potential therapeutic benefit [2]. This proposal seeks to develop a revolutionary new tool for assessing multiple epigenetic modifications, simultaneously and genome-wide, in vanishingly small quantities of material. If successful, this will vastly increase the epigenetic analyses possible in the now-forming Reference Epigenome Mapping Centers. This technology will be of enormous benefit to the discovery mission of these Centers and their impact on public health.