Genome-wide detection of cytosine methylation by single molecule real-time sequencing

S-Methylcytosine (SmC) is an important type of epigenetic modification. Bisulfite sequencing (BS-seq) has limitations, such as severe DNA degradation. Using single molecule real-time sequencing, we developed a methodology to directly examine SmC. This approach holistically examined kinetic signals of a DNA polymerase (including interpulse duration and pulse width) and sequence context for every nucleotide within a measurement window, termed the holistic kinetic (HK) model. The measurement window of each analyzed double-stranded DNA molecule comprised 21 nucleotides with a cytosine in a CpG site in the center. We used amplified DNA (unmethylated) and M.Sssl-treated DNA (methylated) (M.Sssl being a CpG methyltransferase) to train a convolutional neural network. The area under the curve for differentiating methylation states using such samples was up to 0.97. The sensitivity and specificity for genome-wide SmC detection at single-base resolution reached 90% and 94%, respectively. The HK model was then tested on human-mouse hybrid fragments in which each member of the hybrid had a different methylation status. The model was also tested on human genomic DNA molecules extracted from various biological samples, such as buffy coat, placental, and tumoral tissues. The overall methylation levels deduced by the HK model were well correlated with those by BS-seq (r = 0.99; P < 0.0001) and allowed the measurement of allele-specific methylation patterns in imprinted genes. Taken together, this methodology has provided a system for simultaneous genome-wide genetic and epigenetic analyses.

Automated summary

The study developed a methodology for direct detection of S-Methylcytosine, achieving genome-wide SmC detection at single-base resolution in various biological samples with high sensitivity and specificity. It also allowed for the measurement of allele-specific methylation patterns in imprinted genes.