Ultra low-input epigenetic sequencing with combined enzymatic and long-read technologies

PROJECT SUMMARY This project seeks to advance epigenetic sequencing by combining novel enzymatic methods with long-read sequencing technologies, allowing for the accurate resolution of epigenetic and genetic information across extended DNA regions even in sparse, but biologically-important, genomic DNA samples. Traditional epigenetic sequencing has heavily relied on bisulfite sequencing (BS-Seq), which, while effective for identifying 5- methylcytosine (5mC), confounds this base with the opposing and second most common mark 5- hydroxymethylcytosine (5hmC), and degrades DNA, limiting its use across long-ranges and in low-input samples. Moreover, third-generation sequencing platforms, despite their ability to map long DNA fragments and detect modifications directly, require high sample inputs, which constrains their application in critical areas, such as single-cell analyses and circulating cell-free DNA (cfDNA) studies. Our project will focus on overcoming these limitations by seamlessly integrating cutting-edge enzymatic approaches that permit the targeted conversion of cytosine modifications with the capabilities of long-read sequencing to provide a more comprehensive view of the epigenetic landscape. These technologies will be specifically tailored to handle ultra-low input samples efficiently, addressing significant gaps in current methodologies. The project's main goals are two-fold. First, we aim to employ a combination of unnatural cytosine analogs and helicase-deaminase fusions to perform integrated long-read sequencing that preserves both genetic and epigenetic information, enabling the enrichment and precise profiling of DNA modifications (5mC and 5hmC) in cfDNA from cancer patients. This approach will facilitate the detection of cancer-specific modifications and potentially better pinpoint the tissue of origin in unknown cancer samples. Second, we will develop a novel enzyme-based, single-cell barcoding, and amplification strategy combined with long-read sequencing to map 5mC and 5hmC modifications in single-cell epigenomes, particularly focusing on brain tissues. This will allow us to explore cellular heterogeneity and the role of 5hmC in gene regulation within individual cell types. By leveraging our interdisciplinary expertise and innovative methodologies, this project aims to redefine the boundaries of genomic research, providing a more detailed and functional map of the human epigenome. This work will not only enhance our understanding of basic sciences of human development and disease but also support the development of more effective diagnostic tools based on epigenetic markers.