Cytosine, 5-methylcytosine, and 5-hydroxymethylcytosine were identified during translocation of single DNA template strands through a modified Mycobacterium smegmatis porin A (M2MspA) nanopore under control of phi29 DNA polymerase. This identification was based on three consecutive ionic current states that correspond to passage of modified or unmodified CG dinucleotides and their immediate neighbors through the nanopore limiting aperture. To establish quality scores for these calls, we examined ∼3,300 translocation events for 48 distinct DNA constructs. Each experiment analyzed a mixture of cytosine-, 5-methylcytosine-, and 5-hydroxymethylcytosine-bearing DNA strands that contained a marker that independently established the correct cytosine methylation status at the target CG of each molecule tested. To calculate error rates for these calls, we established decision boundaries using a variety of machine-learning methods. These error rates depended upon the identity of the bases immediately 5′ and 3′ of the targeted CG dinucleotide, and ranged from 1.7% to 12.2% for a single-pass read. We estimate that Q40 values (0.01% error rates) for methylation status calls could be achieved by reading single molecules 5-19 times depending upon sequence context.MspA | epigenetics E pigenetic modifications of DNA help regulate gene transcription in biological cells. In mammals, 5-methylcytosine (mC) modification of CG dinucleotides is known to influence development (1, 2) and contribute to human diseases including cancer (3). Other modifications have been detected at carbon 5 of cytosine including 5-hydroxymethylcytosine (hmC) (4), and more recently 5-formylcytosine, and 5-carboxycytosine (5). Physiological roles for hmC in carcinogenesis and embryonic stem cell differentiation have been proposed (6).High-throughput techniques for mC detection are based on bisulfite treatment of genomic DNA (7). In the conventional assay, cytosine (but not mC nor hmC) is converted to uracil (8). Thus, positions not converted to uracil identify cytosines that were modified in the original genomic sequence. In a landmark paper, Lister et al. (9) used this technique to map genome-wide cytosine methylation in human embryonic stem cells and fetal lung fibroblasts at single-nucleotide precision. Recently, bisulfite strategies for discriminating between mC and hmC using the Tet1 enzyme (10) or by chemical modification of hmC (11) have been described.Single-molecule techniques have emerged as possible alternatives to bisulfite treatment for detecting epigenetic modifications of DNA (12). These single-molecule approaches share several useful features including few processing steps before sequence analysis, long reads that routinely exceed several thousand nucleotides, and the ability to read native DNA strands in heterogeneous mixtures. The most advanced of these single-molecule techniques, from Pacific Biosciences, uses fluorescence to detect labeled nucleotide triphosphates during daughter-strand elongation. This elongation is catalyzed by a DNA pol...

Individual DNA molecules can be read at single nucleotide precision using nanopores coupled to processive enzymes. Discrimination among the four canonical bases has been achieved, as has discrimination among cytosine, 5-methylcytosine (mC), and 5-hydroxymethylcytosine (hmC). Two additional modified cytosine bases, 5-carboxylcytosine (caC) and 5-formylcytosine (fC), are produced during enzymatic conversion of hmC to cytosine in mammalian cells. Thus, an accurate picture of the cytosine epigenetic status in target cells should also include these C5-cytosine variants. In the present study, we used a patch clamp amplifier to acquire ionic current traces caused by phi29 DNA polymerase-controlled translocation of DNA templates through the M2MspA pore. Decision boundaries based on three consecutive ionic current states were implemented to call mC, hmC, caC, fC, or cytosine at CG dinucleotides in ∼4400 individual DNA molecules. We found that the percentage of correct base calls for single pass reads ranged from 91.6% to 98.3%. This accuracy depended upon the identity of nearest neighbor bases surrounding the CG dinucleotide.