Single-step deamination sequencing of 5-hydroxymethylcytosine in genomic DNA at single-base resolution

5-Hydroxymethylcytosine (5hmC) is an important epigenetic mark that can regulate gene expression. While some methods were developed to detect 5hmC, direct genome-wide mapping of 5hmC at base resolution are still highly desirable. Herein, we proposed a single-step deamination sequencing (SSD-seq) method for the genome-wide mapping of 5hmC at single-base resolution. This method capitalizes on a screened engineered human apolipoprotein B mRNA-editing catalytic polypeptide-like 3A (A3A) protein to produce differential deamination activity toward cytosine (C), 5-methylcytosine (5mC), and 5hmC. In SSD-seq, an engineered A3A protein (eA3A-v10) can adequately deaminate C and 5mC, but not 5hmC. The original C and 5mC in DNA are deaminated by eA3A-v10 to form uracil (U) and thymine (T), both of which are read as T during sequencing. However, 5hmC is resistant to the deamination by eA3A-v10 and is still read as C during sequencing. Therefore, the remaining C in the sequence reads manifests the original 5hmC. Applying SSD-seq to generate a base-resolution map of 5hmC in human lung tissue, we found that 5hmC was almost entirely confined to CpG dinucleotides. The base-resolution map of 5hmC from human lung tissue generated by SSD-seq correlated strongly with that generated by prior ACE-seq. Taken together, the SSD-seq method is single-step, bisulfite-free and does not require DNA glycosylation or chemical treatment, which offers a valuable tool for the direct and quantitative detection of 5hmC in genomes at single-base resolution. Overall design: Single-base resolution mapping of 5-hydroxymethylcytosine in human normal lung tissue by SSD-seq and ACE-seq. DD-seq/SSD-seq (resolutionSingle-step deamination sequencing) mapping 5hmC at single-base. ACE-seq (APOBEC-Coupled Epigenetic Sequencing): Schutsky, E. K et al. (Nat Biotechnol 2018, 36, 1083-1090). There are two data columns in the 'P5_10X_P5M_10X_10kb_amf.txt': the 5hmC signals from ACE-seq are provided in fileID_P5_10X.merged.hapinfo.txt column the 5hmC signals from SSD-seq are provided in fileID_P5_M_10X.merged.hapinfo.txt column