Phasing single-molecule nano-NOMe-seq reveals chromatin state heterogeneity in the context of transcription and long-range interactions

ABSTRACT A central challenge in molecular biology is determining how 3D chromatin architecture, particularly enhancer-promoter looping and insulating CTCF-mediated interactions, influences gene transcription in individual cells, which has significant implications for healthy and diseased states. To overcome current limitations in imaging and genomic technologies, we developed a cluster-based phasing strategy using long read nano-NOMe-seq to link distinct CTCF binding states— captured at the single molecule level—to the transcriptional status of genes. By stitching partially overlapping long reads and clustering them by shared GpC-accessibility patterns, we stratify CTCF into graded binding states on individual molecules, classify RNA polymerase states at promoters/gene bodies, and infer when spatially separated loci are coordinately activated and occupy loop-competent configurations on the same molecules. When applied to Sox2, Hoxa, and Klf1 regions, cluster-based nano-NOMe-seq phasing reveals how specific topologies bias polymerase behavior and multi-locus activity in ways that bulk assays or locus-engineered imaging cannot fully capture. Competing Interest Statement The authors have declared no competing interest. Footnotes Fixes in Figure 1 (one profile in B and PI formula in C) Figure 5 and 6 (color of anchor validated by ORCA in legend box) Correction in Figure 7 legend (region length)