Emily Madden, PhD: No financial relationships to disclose
Introduction/Rationale: Chromatin structure regulates gene expression, shaping immune cell responses and differentiation. Profiling accessibility, transcription factor binding, and DNA methylation is key to understanding immune regulation. Short-read sequencing (SRS) methods like ATAC-seq and ChIP-seq provide valuable insights but lack resolution and cannot capture multiple chromatin features together, limiting our ability to define their coordinated roles in immune function.
Methods: We optimized Fiber-seq, a multiomic long-read sequencing (LRS) method to simultaneously profile chromatin accessibility and endogenous methylation using LRS. Fiber-seq uses a non-specific DNA methyltransferase to label accessible DNA by creating N6-methyladenosine, a mark rarely found in eukaryotic genomes. This rapid enzymatic incubation also preserves endogenous DNA methylation so that both methylation marks can be detected by direct LRS.
Results: We validated this new method using human lymphoblast cells (K562). Aggregate Fiber-seq accessibility profiles were highly concordant with published ATAC-seq datasets. We confirmed that 6mA-labeling preserved sequencing accuracy and detection of CpG methylation. We also found nucleosome and protein footprints could be inferred from Fiber-seq data. We have validated multiple transcription factor inferred footprints are highly concordant with published CUT&RUN or ChIP-seq datasets. Further, these footprints are captured at a per-molecule and near-base pair resolution, uncovering multiple binding events in close proximity, which is usually obscured by lower resolution SRS-based methods.
Conclusion: Fiber-seq simultaneously profiles chromatin accessibility, DNA methylation, protein footprints, and genetic variation on single molecules at near–base-pair resolution. This integrated view reveals how genetic and epigenetic features interact to regulate gene expression and provides a powerful new framework for dissecting immune cell function and disease mechanisms.
