Direct enzymatic sequencing of 5-methylcytosine at single-base resolution

• Published in: Nature chemical biology Vol. 19; no. 8; pp. 1004 - 1012
• Main Authors: Wang, Tong, Fowler, Johanna M., Liu, Laura, Loo, Christian E., Luo, Meiqi, Schutsky, Emily K., Berríos, Kiara N., DeNizio, Jamie E., Dvorak, Ashley, Downey, Nick, Montermoso, Saira, Pingul, Bianca Y., Nasrallah, MacLean, Gosal, Walraj S., Wu, Hao, Kohli, Rahul M.
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.08.2023; Nature Publishing Group
• Subjects: 5-Methylcytosine; 631/208; 631/92/147; Animals; Biochemical Engineering; Biochemistry; Bioorganic Chemistry; Bisulfite; Cell Biology; Chemistry; Chemistry and Materials Science; Chemistry/Food Science; Cytosine; Deamination; Deoxyribonucleic acid; DNA; DNA - genetics; DNA Methylation; DNA methyltransferase; Genetic testing; Genomes; Localization; Mammals - genetics; Nondestructive testing; Nucleotide sequence; Sequence Analysis, DNA - methods; Substrates
• ISSN: 1552-4450; 1552-4469
• DOI: 10.1038/s41589-023-01318-1

5-methylcytosine (5mC) is the most important DNA modification in mammalian genomes. The ideal method for 5mC localization would be both nondestructive of DNA and direct, without requiring inference based on detection of unmodified cytosines. Here we present direct methylation sequencing (DM-Seq), a bisulfite-free method for profiling 5mC at single-base resolution using nanogram quantities of DNA. DM-Seq employs two key DNA-modifying enzymes: a neomorphic DNA methyltransferase and a DNA deaminase capable of precise discrimination between cytosine modification states. Coupling these activities with deaminase-resistant adapters enables accurate detection of only 5mC via a C-to-T transition in sequencing. By comparison, we uncover a PCR-related underdetection bias with the hybrid enzymatic-chemical TET-assisted pyridine borane sequencing approach. Importantly, we show that DM-Seq, unlike bisulfite sequencing, unmasks prognostically important CpGs in a clinical tumor sample by not confounding 5mC with 5-hydroxymethylcytosine. DM-Seq thus offers an all-enzymatic, nondestructive, faithful and direct method for the reading of 5mC alone. Wang et al. developed a bisulfite-free method termed DM-Seq that leverages an unnatural enzyme–substrate pair coupled with a DNA deaminase to sequence 5-methylcytosine at base resolution in sparse DNA samples, circumventing the limitations of chemical deamination methods.