Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome

Although DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct -Read 6mA sequencing (DR-6mA-seq), an antibody -independent method, to measure 6mA at base resolution. DR-6mA-seq employs a unique mutation -based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through the successful mapping of the well -characterized G(6mA)TC motif in the E. coli DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is generally low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single -base resolution for a comprehensive understanding of DNA 6mA in eukaryotes.