Multiomic Long-Read Sequencing Tools for Chromatin State and Genetic Variation

High-resolution epigenomic insights are vital to inform the discovery of biomarkers and mechanisms in cancer research. Modern short-read sequencing (SRS) methods such as ATAC-seq, WGBS, and CUT&RUN/ChIP-seq have served as workhorses for studying chromatin architecture, but they are fundamentally limited. These methods lack multiomic insights, requiring labor-intensitve, costly, and sample-consuming parallel assays. Moreover, these assays fragment DNA, losing critical information about coordinated regulatory effects, and are blind to repetitive and structurally complex regions of the genome. These limitations constrain progress in understanding gene regulation and translating genetic discoveries into clinical insights. To overcome these limitations, EpiCypher is developing multiomic long-read sequencing (LRS) technologies that enable single-molecule epigenomic profiling. These approaches directly record chromatin features onto native DNA, preserving the intrinsic relationships between chromatin states and genetic variation across individual DNA molecules. Fiber-seq employs an N6-adenine (6mA) methyltransferase (Hia5) to record chromatin accessibility directly onto DNA, while retaining endogenous DNA methylation (5mC). We have streamlined Hia5 manufacturing and labeling workflows, enabling robust single-molecule chromatin accessibility profiling with near base-pair resolution, readily adopted at the bench. Fiber-seq reveals precise transcription factor (TF) binding footprints and cis-regulatory haplotypes as demonstrated in a genetic disease diagnosis (PMID: 40166185), highlighting its potential for variant-to-function discovery in diseases. DAF-seq (Deaminase-Assisted single-molecule chromatin Fiber sequencing), complements this approach by using a dsDNA cytosine deaminase (SsDddA) to encode chromatin accessibility as a mutation maintained through PCR amplification. As a result, DAF-seq can deliver high-resolution chromatin profiles and TF footprints from low-inputs or even single-cells (bioRxiv DOI: 10.1101/2024.11.06.622310). Remarkably, DAF-seq delivers nearly a 7,000-fold improvement in genome coverage compared to leading single-cell ATAC-seq technologies, while operating at a fraction of the cost.Together, Fiber-seq and DAF-seq represent a versatile toolkit: Fiber-seq provides an unbiased multiomic discovery to simultaneously assess chromatin accessibility, DNA methylation, and inferred TF binding genome-wide; while DAF-seq enables targeted chromatin accessibility mapping from low-input samples, resolving heterogeneity at single-molecule resolution. These LRS technologies bridge genetic and chromatin regulatory information on single molecules to reveal disease mechanisms, define drug responses, and drive precision medicine. Citation Format: Lu Sun, James T. Anderson, Connor P. Frasier, Allison R. Hickman, Sabrina R. Hunt, Emily A. Madden, Keith E. Maier, Andrea L. Johnstone, Zu-Wen Sun, Martis W. Cowles, Andrew B. Stergachis, Bryan J. Venters, Michael-Christopher Keogh. Resolving genome regulatory complexity with new multiomic long-read sequencing tools for chromatin state and genetic variation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3225.