SMAC-seq

Epigenetic regulation depends on the interplay between chromatin accessibility and DNA methylation — two layers of information that are typically measured by separate assays on fragmented DNA. Single-molecule accessible chromatin and methylation sequencing (SMAC-Seq) breaks this limitation by combining enzymatic labeling of open chromatin with Oxford Nanopore long-read sequencing to simultaneously profile chromatin accessibility, native DNA methylation, and genetic sequence on individual DNA molecules. CD Genomics offers a comprehensive SMAC-Seq service that delivers kilobase-scale single-molecule epigenetic multi-omics from a single experiment.

Our SMAC-Seq platform uses sequence-nonspecific methyltransferases — EcoGII for m6A labeling of accessible chromatin, with optional M.CviPI (GpC methylation) and M.SssI (CpG methylation) for combined accessibility-methylation analysis. After high-molecular-weight DNA extraction, Nanopore long-read sequencing detects these exogenous modifications alongside endogenous DNA methylation at single-molecule resolution. This approach provides chromatin accessibility at ~3–5 bp theoretical resolution, captures coordinated accessibility across distal regulatory elements up to 100 kb apart, and simultaneously reports native 5-methylcytosine (5mC) patterns — all without fragmentation, bisulfite conversion, or amplification.

Why Choose Our SMAC-Seq Service?

• Single-molecule multi-omics — Simultaneously captures chromatin accessibility, DNA methylation, and genetic variation on individual DNA molecules from one sequencing run
• Amplification-free long reads — Direct detection of enzymatic and endogenous modifications without PCR amplification, preserving native signal accuracy across multi-kilobase reads
• Long-range regulatory coordination — Resolves accessibility states across promoters, enhancers, and insulators on single DNA molecules, enabling correlation analysis at true genomic distances
• Comprehensive bioinformatics — Dedicated pipeline for per-base modification calling, single-molecule chromatin state classification, nucleosome positioning, and transcription factor footprinting

What is SMAC-Seq?

SMAC-Seq (Single-Molecule long-read Accessible Chromatin mapping Sequencing) profiles chromatin accessibility and DNA methylation simultaneously on individual DNA molecules at multi-kilobase scales. The core principle involves treating intact nuclei with sequence-nonspecific DNA methyltransferases that preferentially modify accessible (open) chromatin regions while nucleosome-bound or protein-protected DNA remains unmodified. After high-molecular-weight DNA extraction, Oxford Nanopore long-read sequencing directly detects both the exogenous and endogenous base modifications from the raw ionic current signal, providing single-molecule resolution of chromatin states across multi-kilobase genomic distances.

The key advantage of SMAC-Seq over conventional short-read methods (ATAC-seq, DNase-seq) is its ability to profile accessible chromatin on native DNA molecules without fragmentation. This preserves the long-range connectivity between distal regulatory elements — promoters, enhancers, insulators — enabling analysis of coordinated chromatin states across tens to hundreds of kilobases on individual DNA fibers. SMAC-Seq simultaneously captures endogenous DNA methylation (5mC in CpG context), delivering multi-modal epigenetic profiles from a single experiment. For complementary epigenomic approaches, explore our Epigenetics and Methylation Analysis platform.

Enzyme Variants

m6A-SMAC-Seq (EcoGII only): Uses EcoGII methyltransferase to label accessible chromatin at all adenine residues (N6-methyladenosine / m6A). Ideal for human and mammalian samples where endogenous CpG methylation is already present, as EcoGII labeling does not interfere with 5mC detection. Provides the highest resolution (~3–5 bp), enabling transcription factor footprinting analysis at single-molecule resolution.

m6A-CpG-GpC-SMAC-Seq (EcoGII + M.CviPI + M.SssI): Combines three methyltransferases for simultaneous accessibility profiling and comprehensive methylation analysis. M.CviPI methylates GpC contexts and M.SssI methylates CpG contexts (both producing 5mC). This variant is particularly suitable for organisms with minimal endogenous DNA methylation (yeast, Drosophila) and for studies requiring both exogenous accessibility marks and endogenous methylation detection in a single assay.

Applications of SMAC-Seq

Chromatin Accessibility Profiling at Single-Molecule Resolution

• Genome-wide mapping of open chromatin with multi-kilobase single-molecule reads covering promoters, enhancers, and repetitive regions
• Nucleosome positioning and occupancy analysis on individual DNA fibers without chemical crosslinking or MNase digestion
• Mapping accessible chromatin in complex genomic regions (centromeres, telomeres, rDNA arrays) inaccessible to short-read methods

Transcription Factor Footprinting and Regulatory Element Discovery

• Single-molecule detection of transcription factor binding events via protection from exogenous methylation
• Identification of active transcription factor binding motifs and condition-specific regulatory element usage in development and disease
• Mapping of insulator elements (CTCF) and boundary regions with long-range chromatin state correlation data

Coordinated Long-Range Epigenetic Analysis

• Direct observation of coordinated accessibility between distal enhancers and promoters on individual DNA molecules
• Analysis of mutually exclusive chromatin state usage and chromatin state switching at developmentally regulated loci
• Allele-specific accessibility and methylation analysis on phased haplotypes for investigating imprinting and X-inactivation

Cancer Epigenomics and Allele-Specific Studies

• Comprehensive profiling of tumor-specific chromatin remodeling and aberrant DNA methylation in cancer genomes
• Allele-specific epigenetic analysis at imprinted loci, tumor suppressor genes, and oncogenic fusion breakpoints
• Long-range characterization of epigenetic silencing domains and chromatin state heterogeneity within tumor populations

Developmental Biology and Stem Cell Research

• Single-molecule epigenetic profiling during cellular differentiation and reprogramming using long-read multi-omics approaches
• Analysis of chromatin state transitions at developmental gene loci and imprinted regions across cell lineages
• Haplotype-resolved epigenomic analysis for investigating allele-specific regulation in hybrid or cross-species systems

Bioinformatics Analysis

Sample Requirements for SMAC-Seq

For detailed instructions on sample preparation and shipping, please refer to our Sample Submission Guidelines.

Case Study: MAGNIFIER — Data-Adaptive SMAC-Seq Chromatin Accessibility Detection

Tu K, Li X, Zhang Q, Huang W, Xie D. A data-adaptive method for detecting exogenous methyltransferase-accessible chromatin using nanopore sequencing. Bioinformatics. 2024;40(5):btae206. DOI: 10.1093/bioinformatics/btae206. (CC BY 4.0)

1. Background

SMAC-Seq data analysis faces three key challenges: endogenous DNA methylation can be confused with exogenous labeling signals, non-open-chromatin-specific exogenous methylation introduces noise, and nanopore base-calling errors affect per-base modification calls. Existing methods lack data-adaptive strategies to distinguish true chromatin accessibility signals from these confounding factors, particularly in repetitive genomic regions where short-read methods are blind. The authors developed MAGNIFIER (Methyltransferase Accessible Genome Region Finder) to address these challenges using a hierarchical latent variable model.

2. Methods

MAGNIFIER employs a hierarchical latent variable model that integrates co-methylation scores across neighboring sites to reduce noise, predicts locus-specific null distributions to account for regional background methylation, determines per-read modification status (Z) at each candidate base using data-adaptive thresholds, and computes chromatin accessibility scores from the aggregated per-read status. The model was trained and validated on SMAC-Seq data (EcoGII-treated nuclei followed by Nanopore sequencing) from human K562 and GM12878 cell lines, with ATAC-seq and DNase-seq data used as ground truth for performance benchmarking.

3. Results

Figure 1. MAGNIFIER workflow for SMAC-Seq data analysis. (A) Overview of the hierarchical latent variable model describing the relationship between exogenous methylation, endogenous methylation, and chromatin accessibility. (B) Calculation of co-methylation score to quantify coordinated modification across neighboring sites. (C) Prediction of null distribution for each candidate base site using a data-adaptive approach. (D) Identification of locus-specific stage cutoff and per-read modification status (Z) for each candidate base showing the genomic structure of exogenous methylation signal. (E) Prediction of per-site exogenous modification stage based on aggregated per-read status Z. (F) Calculation of chromatin accessibility scores from exogenous modification calls. Adapted from Tu K, et al. Bioinformatics. 2024;40(5):btae206. (CC BY 4.0)

Key Findings

• Improved detection accuracy — MAGNIFIER outperformed existing base-calling methods for open chromatin detection, with significantly higher AUC in ROC analysis across K562 and GM12878 cell lines
• Repetitive region accessibility — Uniquely detected open chromatin in repetitive genomic regions (including Alu elements) that are invisible to short-read ATAC-seq, revealing accessible chromatin within repeat sequences linked to alternative transcription start and end sites
• Specificity through data-adaptive modeling — The hierarchical latent variable model with per-locus null distributions effectively distinguished true chromatin accessibility from endogenous methylation interference and base-calling noise
• Method validation across cell types — Demonstrated robust performance across multiple human cell lines (K562, GM12878), with open chromatin region calls validated against independent ATAC-seq and DNase-seq datasets
• Novel transcript discovery — Open accessible Alu elements detected by MAGNIFIER were associated with non-classical transcript isoforms, suggesting SMAC-Seq can reveal regulatory elements linked to alternative gene regulation

4. Conclusions

The MAGNIFIER study demonstrates that dedicated computational approaches significantly enhance the value of SMAC-Seq data by accurately detecting chromatin accessibility across all genomic contexts, including repetitive regions inaccessible to short-read methods. By addressing the unique analytical challenges of exogenous methyltransferase labeling combined with nanopore long-read sequencing, MAGNIFIER provides a robust framework for extracting biological insights from SMAC-Seq experiments and highlights the technology's potential for comprehensive epigenomic analysis beyond the limits of traditional approaches.

Demo Data and Deliverable Examples

Deliverable 1 — Single-molecule chromatin accessibility tracks
IGV genome browser view showing per-molecule modification patterns across a gene locus, with open chromatin (methylated) and closed/protected (unmethylated) segments color-coded along each aligned read. Individual DNA molecules are displayed as parallel tracks, revealing heterogeneity in chromatin states across the cell population.

Deliverable 2 — Per-base modification frequency tracks
Genome browser tracks for both exogenous (6mA accessibility) and endogenous (5mC methylation) modifications, displayed as continuous frequency profiles across genomic coordinates. Aggregate tracks are directly comparable to short-read ATAC-seq and WGBS data for validation and integration.

Deliverable 3 — Single-molecule chromatin state classification
Color-coded molecule-level view distinguishing open chromatin (red), nucleosome-bound (blue), and transcription factor-protected (green) regions along each DNA molecule, with aggregate state enrichment plots at annotated regulatory elements.

Deliverable 4 — Transcription factor footprinting analysis
Aggregated methylation protection profiles at transcription factor binding motifs, showing characteristic dip patterns at occupied sites. Condition-specific footprint depth comparisons reveal differential transcription factor occupancy across experimental conditions.

Deliverable 5 — Long-range coordinated accessibility analysis
Correlation matrices and molecule-level views showing coordinated open/closed chromatin states between distal enhancer-promoter pairs, enabling identification of regulatory interactions maintained on individual DNA molecules.