SMAC: identifying DNA N⁶-methyladenine (6mA) at the single-molecule level using SMRT CCS data

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 CCS data from the Sequel II system. It is an automated method that streamlines the entire workflow by packaging both existing software and built-in script, with support for user-defined parameters to allow easy adaptation for various studies. This algorithm utilizes the statistical distribution characteristics of enzyme kinetic indicators to identify 6mA of each DNA molecule, rather than relying on a fixed cutoff, which significantly improves accuracy at the single-nucleotide and single-molecule level. SMAC is a powerful new tool that enables de novo detection of 6mA and empowers investigation of its functions in modulating physiological processes.