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. Overall design: 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.

亚硫酸氢盐处理会对DNA造成损伤，导致DNA片段化并丢失长程甲基化信息。为克服亚硫酸氢盐处理DNA的这一局限，我们将一种名为EM-seq（Enzymatic Methyl-seq，酶法甲基化测序）的新型酶促脱氨方法应用于长程测序技术。我们的方法命名为LR-EM-seq（Long Range Enzymatic Methyl-seq，长程酶法甲基化测序），可保留DNA完整性，实现数千碱基对基因组DNA区域内5-甲基胞嘧啶（5-mC）与5-羟甲基胞嘧啶（5-hmC）的长程甲基化谱分析。
将该方法应用于已知的差异甲基化区域（DMR, Differentially Methylated Regions）时，LR-EM-seq可实现超过5 kb的相位分析，所得到的差异甲基化区域比以往报道的范围更广、定义更清晰。
总体实验设计：我们将一种用于5-胞嘧啶修饰检测的新型酶促脱氨方法适配至长读长测序技术（如PacBio单分子实时测序（PacBio SMRT sequencing）与牛津纳米孔测序（Oxford Nanopore sequencing）），以对长DNA分子的5-甲基胞嘧啶与5-羟甲基胞嘧啶进行相位分析。本文中命名为长读长EM-seq（LR-EM-seq）的最终方法，在通过APOBEC3A介导的酶促脱氨与长片段测序样本制备之前，先对5-甲基胞嘧啶和/或5-羟甲基胞嘧啶进行选择性酶保护，从而实现碱基分辨率下5-甲基胞嘧啶与5-羟甲基胞嘧啶的精准谱分析。DNA完整性的保留使得我们能够对数千碱基对的基因组DNA进行位点特异性扩增，并实现5-甲基胞嘧啶与5-羟甲基胞嘧啶的分子分辨率长程相位分析。