e29 Nucleic Acids Research, 2012, Vol. 40, No. 4 durations (IPDs) were measured as previously described (28) for all pulses aligned to each position in the reference sequence. For each position, the distribution of IPDs was compared between the native and WGA samples separate- ly for the forward and reverse strands. The comparison was made using baseline-corrected IPD ratios. Baseline correction was applied by dividing the IPD mean for each position by the mean of mean IPDs over all positions in the plasmid, excluding those positions where the known methyltransferase recognition motif was detected and a window of 6 bases in each direction around such positions. This correction was applied to both the native sample IPD mean and the WGA control mean before obtaining the ratio of the two. In addition, 5% of outlier values were trimmed from both sides of the IPD distribution at each position before taking the ratio. IPD ratio plots were visualized using Circos (33). The uncertainty in the IPD ratio was quantiﬁed by calculating the standard error of the mean of the IPD ratio using the delta method: S E 1 2 rﬃ1ﬃsﬃsﬃﬃﬃﬃﬃﬃﬃ2ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ n 12 412+ s2221 where m1 and m2 are the average IPD values of the native and control, s1 and s2 are their standard deviations and n is the lower sequencing coverage of the two samples. The relationship between this standard error and the sequencing fold coverage is shown in Supplementary Figure S1 for three different methyltransferases (M.EcoKDam, M.EsaLHCI, M.Sau3AI). RESULTS SMRT DNA sequencing of plasmids containing MTase-mediated methylation Plasmids containing cloned DNA-methyltransferase genes were expressed in an E. coli strain that otherwise lacked endogenous methyltransferase activities. The expressed methyltransferase acts to modify a speciﬁc recognition sequence by adding a methyl group to individual bases forming m6A, m4C or m5C. The methyltransferase will modify both the genomic and plasmid DNA. Plasmid DNAs containing the modiﬁcations were isolated and converted into SMRTbell templates to facilitate sequencing (30). Plasmids were also subjected to whole-genome ampliﬁcation (WGA) to create a control DNA template lacking any DNA base modiﬁcations. Native and control samples were subjected to SMRT DNA sequencing (Figure 1). SMRT DNA Sequencing involves monitoring an individual DNA polymerase while it is replicating the input DNA, employing phospholinked nucleotides with different ﬂuorophores for each of the four bases to produce ﬂuorescence pulses at each incorporation step (29,32). As the DNA polymer- ase is sensitive to even subtle perturbations in the DNA template, it is possible to detect the presence of a methylated base by analyzing the polymerase kinetics as it moves across the DNA (28). This is manifested by the polymerase slowing down in a predictable manner upon PAGE 4 OF 12 Figure 1. Principle of characterizing MTase speciﬁcities by SMRT DNA sequencing. In SMRT sequencing, single molecules of an engin- eered phi29-based polymerase are monitored in real-time, using ﬂuorescently-labeled phospholinked nucleotides, as they synthesize a complementary strand from the DNA template strand that contains methylated bases. The timing of ﬂuorescence pulses corresponding to nucleotide incorporations is analyzed and compared with a control template lacking methylated bases. The kinetics of DNA synthesis is affected by the presence of a methylated base in the template, e.g. by increasing the time prior to nucleotide binding across the methylated base, resulting in an increased IPD. The ratio of IPDs between native and control samples for each template position yield kinetic signatures for identifying the presence of methylated bases in the DNA template, thus deﬁning MTase speciﬁcities. encountering a base modiﬁcation relative to the speed of copying the same non-modiﬁed control sequence. Thus, the time between ﬂuorescent pulses (interpulse duration or IPD) is longer for a DNA template that contains a methylated base than for one that does not (28). One