Multiomic genomic mapping with long read sequencing

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PROJECT SUMMARY Genomic mapping of histone post-translational modifications (PTMs), chromatin-associated proteins (CAPs), and DNA methylation (DNAme) is a powerful approach for biomedical research and drug development. Current genomics assays (e.g. ChIP-seq, CUT&RUN) rely on second generation short-read sequencing (SRS), wherein short reads (<500bp) limit the ability to a) analyze concordance of epigenomic features on a single DNA molecule and b) map to repetitive regions of the genome. Third generation long-read sequencing (LRS) platforms are capable of sequencing long reads (>10kb, even >100kb) from a single molecule, and are poised to revolutionize genomics by overcoming the significant limitations of SRS. By preserving long stretches of DNA, LRS allows relationships between features on a single molecule to be used to resolve heterogeneity within mixed populations. This is highly relevant for clinical applications, as it enables analysis of signatures of specific cells within a sample without the need for single cell assays (which generate very sparse data). Further, sequencing of long reads allows mapping to challenging and repetitive regions of the genome, which were previously “unmappable” with SRS. Development of epigenetic mapping assays that use LRS provides an unprecedented opportunity to decipher the chromatin landscape of cells within mixed populations, including within previously unmappable genomic regions. However, assays to measure epigenetic elements using LRS are lacking. Here, EpiCypher is collaborating with LRS expert Dr. Winston Timp at Johns Hopkins University to develop CUTANA-LRS, a first-in-class multiomics assay platform that leverages LRS to simultaneously profile histone PTMs or CAPs and DNAme in a single assay. The innovation of CUTANA-LRS is the development of a proprietary, nondestructive approach for epigenomic mapping that leverages a novel DNA methyltransferase fusion protein to label chromatin features of interest. This approach was inspired by related immunotethering- based approaches for genomic mapping that EpiCypher is developing and commercializing (e.g. CUT&RUN). In CUTANA-LRS, DNA molecules are labeled and preserved intact for LRS, which will allow resolution of heterogeneity within / between data types, and will provide access to previously unmappable genomic regions. Together, these advances will provide a pathway to better understand mechanisms of gene regulation and transcriptional response, including in the context of human disease. In Aim 1, we will optimize CUTANA-LRS and map multiple targets, including within challenging regions, while also profiling native DNAme. In Aim 2, we will rigorously develop CUTANA-LRS by optimizing robust protocols across diverse targets, inputs, sequencing platforms, and incorporate a targeted enrichment approach. In Aim 3, we will prepare for commercial launch of CUTANA-LRS, develop automated protocols, perform external validation, and demonstrate a clinical application. This work will establish CUTANA-LRS as a revolutionary platform for mapping and deciphering the relationships between multiple types of chromatin features with access to previously “unmappable” regions.