Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5- hydroxymethylcytosine at single base resolution

Bisulfite treatment damages the DNA leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite treated DNA we applied a new enzymatic deamination approach, termed EM-seq (Enzymatic Methyl-seq) to long-range sequencing technologies. Our methodology, named LR-EM-seq (Long Range Enzymatic Methyl-seq) preserves the integrity of DNA allowing long-range methylation profiling of 5-mC and 5-hmC) over several kilobases of genomic DNA. Applied to known differentially methylated regions (DMR), LR-EM-seq achieves phasing of over 5 kb resulting in much broader and better defined DMRs than previously reported. We adapted a novel enzymatic deamination method for 5-cytosine modification detection to long read sequencing technologies (such as PacBio SMRT sequencding and Oxford Nanopore sequencing) to phase 5mC and 5hmC of long molecules. The resulting method, termed herein Long-Read-EM-seq (LR-EM-seq), utilizes the selective enzymatic protection of 5mC and/or 5hmC prior to enzymatic deamination by APOBEC3A and large fragment sequencing sample preparation to accurately profile both 5-mC and 5-hmC at base resolution. The preservation of DNA integrity allows the locus-specific amplification of several kb of genomic DNA and the long-range phasing at molecular resolution of 5-mC and 5-hmC.