Mapping and characterizing N6-methyladenine in eukaryotic genomes using single-molecule real-time sequencing

Abstract

N6-Methyladenine (m⁶dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m⁶dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m⁶dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m⁶dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m⁶dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m⁶dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m⁶dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m⁶dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m⁶dA events in eukaryotic genomes.