Abstract 7026: Multi-omic genomic mapping with long read sequencing
Authors: Venters Bryan J. , Hook Paul W. , Kumary Vishnu S. , Hickman Alli R. , Anderson James T. , Vaidya Anup , Ezell Ryan J. , Burg Jonathan M. , Sun Zu-Wen , Cowles Martis W. , Timp Winston , Keogh Michael-Christopher 
Published: March 2024

Abstract
Gene transcription is regulated by the complex interplay between histone post-translational modifications (PTMs), chromatin associated proteins (CAPs), and DNA methylation (DNAme). Mapping their genomic locations and examining the relationships between these chromatin elements is a powerful approach to decipher mechanisms of disease, thereby enabling discovery of novel biomarkers and therapeutics. Leading epigenomic mapping technologies (e.g., ChIP-seq, CUT&RUN) rely upon DNA fragmentation to isolate regions of interest for sequencing on short read platforms (e.g., Illumina). This strategy leads to substantial loss of contextual information regarding the surrounding DNA, precluding the identification of multiple co-occurring epigenomic features on a single DNA molecule. By contrast, long-read sequencing (LRS) platforms are capable of sequencing very long reads from a single molecule (typically &gt;10kb), allowing relationships between features on a single molecule to be used to resolve heterogeneity within mixed populations. Here we report a robust multi-omic method that leverages LRS to simultaneously profile histone PTMs (or CAPs), DNAme, and parental haplotype in a single assay. This nondestructive, epigenomic mapping approach leverages a novel DNA methyltransferase fusion protein (pAG-M.EcoGII) to label DNA underneath antibody-targeted chromatin features, thereby marking sites of interest while preserving DNA molecules intact for LRS. Inspired by our work with state-of-the-art immunotethering-based approaches (CUT&RUN/CUT&Tag), nuclei are bound to magnetic beads to streamline and automate sample processing. Next, adenosines nearby antibody-targeted chromatin features are methylated with pAG-M.EcoGII, which are then directly read from genomic DNA using Oxford Nanopore Technologies or Pacific Biosciences LRS platforms. To determine the capabilities and limitations of this assay, we tested multiple chromatin targets in various cell lines. Importantly, this method is highly reproducible across biological replicates, and highly concordant with orthogonal SRS assays (e.g., CUT&RUN). Further, we showed that this method is a true multi-omic approach by simultaneously profiling histone PTMs, native DNAme (5mC), and parental single-nucleotide variants from single DNA molecules within a single reaction. Finally, this workflow preserves chromatin integrity for LRS, revealing heterogeneity (e.g., haplotype or paternal origin) within/between data types and providing access to previously unmappable genomic regions (e.g., centromeres).
Citation Format: Bryan J. Venters, Paul W. Hook, Vishnu S. Kumary, Alli R. Hickman, James T. Anderson, Anup Vaidya, Ryan J. Ezell, Jonathan M. Burg, Zu-Wen Sun, Martis W. Cowles, Winston Timp, Michael-Christopher Keogh. Multi-omic genomic mapping with long read sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7026.