Direct detection of DNA methylation during single-molecule, real-time sequencing

We describe the direct detection of DNA methylation, without bisulfite conversion, through single-molecule real-time (SMRT) sequencing. In SMRT sequencing, DNA polymerases catalyze the incorporation of fluorescently labeled nucleotides into complementary nucleic acid strands. The arrival times and durations of the resulting fluorescence pulses yield information about polymerase kinetics and allow direct detection of modified nucleotides in the DNA template, including N6-methyladenosine, 5-methylcytosine, and 5-hydroxymethylcytosine. Measurement of polymerase kinetics is an intrinsic part of SMRT sequencing and does not adversely affect determination of the primary DNA sequence. The various modifications affect polymerase kinetics differently, allowing discrimination between them. We utilize these kinetic signatures to identify adenosine methylation in genomic samples and show that, in combination with circular consensus sequencing, they can enable single-molecule identification of epigenetic modifications with base-pair resolution. This method is amenable to long read lengths and will likely enable mapping of methylation patterns within even highly repetitive genomic regions.