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N6-Methyladenine (m 6 dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m 6 dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m 6 dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m 6 dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m 6 dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m 6 dA 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 m 6 dA 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 m 6 dA 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 m 6 dA events in eukaryotic genomes.

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