loading
loading
NIH grant 1R44AI197362-01: Scalable, single-molecule and single-cell chromatin accessibility profiling in primary immune cells
Institute: {'code': 'AI', 'abbreviation': 'NIAID', 'name': 'National Institute of Allergy and Infectious Diseases', 'admin_org_id': '12500003', 'admin_funding_url': ''}. PI: Bryan J Venters. Award: $999268.
Start: 2026-08-21T00:00:00.
PROJECT SUMMARY
Chromatin structure dictates which genes and regulatory elements are active and which genomic
elements are silenced through accessibility. Profiling accessible regulatory elements and the proteins that bind
them is critical for understanding the functions and developmental fate of immune cells. Popular assays to study
chromatin accessibility use short-read sequencing (SRS). Long-read sequencing (LRS) platforms can directly
sequence DNA, generating reads >10kb in length and preserving relationships between sequence and
epigenomic features of single molecules. These long reads can be used to resolve heterogeneity through
analysis of cell specific signatures in mixed cell populations. LRS-based accessibility assays provide an
unprecedented opportunity to decipher the chromatin structure of circulating immune cells or cells in complex
tissues like lymph nodes. Recently, novel LRS-compatible chromatin accessibility assays have been developed.
However, these approaches require direct sequencing of the modified DNA, which requires large cell numbers
and sequencing depth, limiting application to rare cell populations and single cells (SCs). An amplification-
compatible LRS chromatin stenciling method would enable analysis of haplotype-specific chromatin structure at
SC resolution with coverage far exceeding all existing SC epigenomic technologies.
Here, EpiCypher is collaborating with Dr. Andrew Stergachis (University of Washington) to develop
DAF-seq, an amplification-compatible multiomic LRS profiling assay to resolve haplotype phased sequence and
chromatin accessibility of single molecules. In this approach, nuclei are treated with a non-specific deaminase
to deaminate accessible cytidine residues, thereby ‘stenciling’ the chromatin accessibility onto individual fibers
during DNA amplification, followed by LRS. With DAF-seq, we observe single-molecule protein occupancy within
broader accessible chromatin regions. Further, because the accessibility information is retained during
amplification, DAF-seq is ideal for samples with low cell numbers, even single cells. In Phase I, we developed a
targeted DAF-seq workflow that provides a ~200,000-fold enrichment relative to leading genome-wide LRS-
based methods. We leveraged this enrichment to identify strand-specific protein footprinting at a well-
characterized promoter. Finally, we developed a single cell DAF-seq workflow, generating the first whole genome
accessibility profile from a single cell, increasing genome coverage 7,000x vs. current scATAC-seq assays using
SRS. In Phase II, we will benchmark our non-specific deaminase against an additional deaminase and develop
robust DAF-seq protocols using multiple immune cell types (Aim 1). Following, we will optimize enrichment of
immune-specific genomic loci and validate DAF-seq analysis at these loci to enable deconvolution of mixed cell
populations (Aim 2). Finally, we will prepare for commercialization by assembling DAF-seq beta kits, launching
a web-based bioinformatics portal, and demonstrating the application of DAF-seq to profile full-length haplotype-
specific chromatin accessibility and protein occupancy from single cells (Aim 3).document fetched 2026-08-30 02:46