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Abstract DNA modifications, such as N6-methyladenine (6mA), play important roles in various processes in eukaryotes. Single-molecule, real-time (SMRT) sequencing enables the direct detection of DNA modifications without requiring special sample preparation. However, most SMRT-based studies of 6mA rely on ensemble-level consensus by combining multiple reads covering the same genomic position, which misses the single-molecule heterogeneity. While recent methods have aimed at single-molecule level detection of 6mA, limitations in sequencing platforms, resolution, accuracy, and usability restrict their application in comprehensive epigenetic studies. Here, we present SMAC (single-molecule 6mA analysis of CCS reads), a novel framework for accurately detecting 6mA at the single-molecule level using SMRT circular consensus sequencing (CCS) data from the Sequel II system. It is an automated method that streamlines the entire workflow by packaging both existing softwares and built-in scripts, with user-defined parameters to allow easy adaptation for various studies. By utilizing the statistical distribution characteristics of enzyme kinetic indicators on single DNA molecules rather than a fixed cutoff, SMAC significantly improves 6mA detection accuracy at the single-nucleotide and single-molecule levels. It simplifies analysis by providing comprehensive information, including quality control, statistical analysis, and site visualization, directly from raw sequencing data. SMAC is a powerful new tool that enables de novo detection of 6mA and empowers investigation of its functions in modulating physiological processes.
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This content will become publicly available on May 21, 2026
Revealing long-range heterogeneous organization of nucleoproteins with 6mA footprinting by ipdTrimming
Enabled by long-read sequencing technologies, particularly Single Molecule, Real-Time sequencing, N6-methyladenine (6mA) footprinting is a transformative methodology for revealing the heterogenous and dynamic distribution of nucleosomes and other DNA-binding proteins. Here, we present ipdTrimming, a novel 6mA-calling pipeline that outperforms existing tools in both computational efficiency and accuracy. Utilizing this optimized experimental and computational framework, we are able to map nucleosome positioning and transcription factor occupancy in nuclear DNA and establish high-resolution, long-range binding events in mitochondrial DNA. Our study highlights the potential of 6mA footprinting to capture coordinated nucleoprotein binding and to unravel epigenetic heterogeneity.
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- Award ID(s):
- 2435178
- PAR ID:
- 10653711
- Publisher / Repository:
- BioMed Central
- Date Published:
- Journal Name:
- Genome Biology
- Volume:
- 26
- Issue:
- 1
- ISSN:
- 1474-760X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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