e29 Nucleic Acids Research, 2012, Vol. 40, No. 4 PAGE 6 OF 12 A B Figure 3. MTase speciﬁcities determined from SMRT sequencing. The sequence context 50-GTAC-30 is methylated by (A) M.CviQI (m6A) and (B) M.RsaI (m4C). The left panel shows IPD ratio data for both strands over the entire plasmid, with the inner and outer circles representing the reverse and forward DNA template strands, respectively. Template positions are indicated by the numbered track middle circle, with blue markers denoting occurrences of MTase target sequence contexts. The right panels show IPD ratios for two representative template positions containing the target context (bold letters); the methylated base is highlighted in red. Error bars represent the standard error of the ratio of means and is calculated as described in the ‘Materials and Methods’ section. Figure S3). For the sequence context 50-GATC-30, MTases exist that can impart all three methylation types: M.EcoKDam (m6A), M.EsaLHCI (m4C) and M.Sau3AI (m5C) (Figure 4). For the dam MTase in E. coli (M.EcoKDam), we obtained data consistent with previously observed kinetic signals for m6A (28) (Figure 4A), showing large IPD ratios at m6A template positions. In addition, we observed a secondary peak at the +5 position relative to the modiﬁed base, likely due to the intimate contact of the DNA polymerase with the nascent double-stranded DNA, and its dynamic sensitivity to slight structural perturbations caused by the presence of the methyl group (28,34). Such secondary peaks enhance the ability to detect and discriminate different base modiﬁcations. Figure 4B shows the methylation activity of M.EsaLHCI that adds a methyl group to the N4 position of cytosine in the 50-GATC-30 sequence context. Encountering m4C DNA base modiﬁcations in the template, the DNA polymerase slows signiﬁcantly when incorporating a G nucleotide opposite the m4C base, re- sulting in IPDs at m4C positions 3- to 5-fold higher for sequences with 4mC compared with unmodiﬁed control sequences. To our knowledge, this study represents the ﬁrst demonstration of directly sequencing the m4C base modiﬁcation. The third common base modiﬁcation in bacteria, m5C, is imparted on the 50-GATC-30 sequence context by the MTase M.Sau3AI (Figure 4C). Whereas the methyl groups in m6A and m4C are directly involved in base pairing, the m5C methyl group is not and is instead pos- itioned in the major groove of the nascent double-stranded DNA which has few direct contacts with the DNA poly- merase. As a result, m5C modiﬁcations cause more subtle perturbations of the DNA which in turn result in smaller effects on the DNA polymerase dynamics (28). While under the current sequencing conditions, the IPD ratios are therefore smaller and spread across multiple bases sur- rounding the modiﬁcation, the majority of m5C positions