Project Summary/Abstract DNA cytosine methylation (5-methylcytosine or 5mC) is a key epigenetic modification in the regulation of human gene expression. It plays critical roles in suppressing transcription of a large portion of human genome, including repetitive elements. 5mC can be reversed through oxidation by the human TET family enzymes, which utilize dioxygen to sequentially oxidize 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and finally 5-carboxycytosine (5caC). Both 5fC and 5caC can be recognized and excised by human thymine DNA glycosylase (TDG), followed by base excision repair (BER) to replace the modified cytosine with a normal cytosine, in an active demethylation process. Cell-type specific DNA methylation has been studied and applied as a biomarker for disease diagnosis and prognosis. Antibody-based MeDIP immunoprecipitation followed by high-throughput sequencing is a common method for genome-wide mapping of DNA 5mC distribution, but it is not quantitative and requires a significant amount of input material. Bisulfite sequencing is the gold standard approach, widely applied in quantitative 5mC sequencing; however, whole-genome bisulfite sequencing is expensive and causes severe DNA degradation. To overcome these critical barriers to progress, we propose to develop new methods that combine 5mC enrichment with quantitative 5mC sequencing, using limited starting material. The potential application of these new methods in circulating cell-free DNA (cfDNA) analyses stands to potentially revolutionize human disease diagnosis and prognosis. Whereas the 5mC modification suppress activation of a majority of human genome, studies from us and others revealed that 5hmC marks active loci. We previously developed robust procedures to map 5hmC genome-wide using 1,000 cells. However, quantitative methods that determine the presence and stoichiometry of 5hmC at the single-cell level are still lacking. Our ongoing efforts to overcome this challenge recently led to a chemical solution that yields base-resolution 5hmC information with modification stoichiometry, using limited input material. In this renewal, we propose base resolution sequencing of 5hmC at the single-cell level, enabling the detection of intercellular differences that are otherwise missed, including the epigenome remodeling that underlies the initiation of cell fates during early embryogenesis. The success of this program will provide new methods for 5mC and 5hmC mapping to enable breakthrough discoveries in both basic research and clinical applications.