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

Abstract

5-Methylcytosine (5mC) 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 5mC. 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.SssI-treated DNA (methylated) (M.SssI 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 5mC 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.

Application of long-read sequencing to the detection of structural variants in human cancer genomes

“…2) For the PacBio sequencer, Tse et al developed a method to detect 5 mCs using SMRT sequencers [75] . This algorithm is based on the difference in inter-pulse duration and pulse width between methylated and unmethylated cytosine.…”

Rapid identification of methylase specificity (RIMS-seq) jointly identifies methylated motifs and generates shotgun sequencing of bacterial genomes

“…Thus, PacBio sequencing misses the m5C methylase activities ( 2 ) unless the 5-methylcytosine detection is enhanced by treating the library with Ten-eleven translocation enzyme ( 6 ). A recent study uses a holistic kinetic model to identify m5C using PacBio reads ( 7 ). Nonetheless, methylation can only be identified in CpG context, restricting the use of this approach to organisms such as human, for which methylation is almost exclusively in CpG sites.…”

Hybrid genome de novo assembly with methylome analysis of the anaerobic thermophilic subsurface bacterium Thermanaerosceptrum fracticalcis strain DRI-13T

“…Sequencing technologies such as Oxford Nanopore technology (ONT) single-molecule real-time (SMRT), whole genome bisulfite sequencing (WGBS), or NEBNext Enzymatic Methyl-seq platforms have made available mapping three of the dominant features in prokaryotic methylomes [18][19][20]. No one method is the perfect tool, biases towards low CG content, over representation of methylation fraction, precision, organism of study, and indels can be found to varying degrees within each platform [21][22][23][24][25][26]. The three typically studied genomic methylation forms are N6-methyladenine (6 mA), N4methylcytosine (4mC), and 5-methylcytosine (5mC) [27,28].…”