PROJECT SUMMARY Chromatin structure can directly regulate gene transcription by local nucleosome positioning / accessibility. Chromatin accessibility assays thus provide a powerful insight to transcriptional activity. The ability to compare chromatin accessibility in healthy and diseased tissue is a major drive: changes in this landscape are associated with a range of human pathologies including cancer, neurological disorders, and aging. However, current chromatin accessibility assays (e.g. ATAC-seq) lack compatibility with both fixed and native (i.e. unfixed) samples. Moreover, it remains challenging to normalize samples for cross-study comparisons, which significantly limits the development of epigenetics-focused drugs and undermines their future clinical dissection (e.g. biomarker development). Here, EpiCypher is partnering with New England Biolabs (NEB) to commercialize UniNicE-seqTM (Universal Nicking Enzyme Assisted Sequencing), a breakthrough chromatin accessibility platform. In contrast to current technologies, UniNicE-seq is fully compatible with native and fixed sample workflows, as well as being highly sensitive and requiring significantly less sequencing depth (>10-fold vs. ATAC-seq). The innovation of this technology is the application of DNA nicking and polymerase enzymes to incorporate biotin-labeled nucleotides into accessible chromatin regions for subsequent DNA purification, library sequencing, and genomic mapping. We have successfully used this approach to reliably generate high quality chromatin accessibility maps in both fixed and native cells. Importantly, these datasets corroborate those generated by current approaches (ATAC-seq and DNase-seq), demonstrating strong proof of concept for UniNicE-seq. Here, our goal is to commercialize UniNicE-seq kits and assay services. EpiCypher is an industry leader in the development of spike-in controls for epigenetics-focused genomic analyses. In Aim 1, we will leverage this expertise to develop recombinant DNA-based spike-in controls for quantitative UniNicE-seq assay normalization. This approach is essential to standardize assay methodology and for reliable cross-sample comparisons. In Aim 2, we will develop and rigorously validate our fully quantitative UniNicE-seq assays in a range of cell and tissue types, using both native and fixed sample workflows. This Aim will also include the development of user-friendly bioinformatic tooling to perform “one-click” analyses of UniNicE-seq samples (including sample normalization and comparisons). In Aim 3, we will develop and validate UniNicE-seq beta kits for commercial launch and end-to-end assay services. Together, these Aims will provide key reagents, methods, and application data as we begin to market our UniNicE-seq kits and end-to-end contract research services for chromatin research and drug discovery.

PROJECT NARRATIVE Chromatin accessibility is a hallmark of active gene transcription and altered in a range of conditions, from cancer to aging. Current chromatin accessibility assays are limited by their ability to work with both fixed and native (i.e. unfixed) samples. Moreover, these assays lack controls for reliable cross-sample comparisons. Here, EpiCypher is partnering with New England Biolabs (NEB) to commercialize UniNicE-seqTM (Universal Nicking Enzyme Assisted Sequencing), a breakthrough quantitative chromatin accessibility assay platform. In contrast to current technologies, UniNicE-seq is fully compatible with native and fixed sample workflows, highly sensitive, and truly quantitative. This innovative and high impact technology will have broad application in the fields of chromatin research and epigenetics-focused drug development.