e29 Nucleic Acids Research, 2012, Vol. 40, No. 4 could be distinguished from unmodiﬁed cytosine based on polymerase kinetics, with each of the MTase target sites showing IPD ratio peaks above background. Determination of methylation patterns of DNA MTases with unknown speciﬁcities We applied our method to determine the methylation patterns of several DNA MTases for which the position and/or type of methylation is not known (Figure 5). The ﬁrst example, M.Tsp509I, was selected from the category of MTases for which the target sequence context and the type of methylation was available, but the exact site of methylation was undetermined. Taking advantage of the base resolution of methylation detection during SMRT sequencing, we determined that it is the inner adenine base of the sequence context 50-AATT-30 that was methylated to m6A (Figure 5A), as the IPD ratio peak was observed at this position, as well as the secondary peak at position +5 (see above). The second example of determining the speciﬁcity of an MTase was from the category of MTases where the target sequence context was available, but both the type and position of methylation were unknown. We sequenced a plasmid expressing M.AatII (Figure 5B), known to methy- late the target sequence 50-GACGTC-30 either at m4C or m6A, and determined that this enzyme methylates the adenine, but not the cytosines, again directly visible from the strong signals at the A position and the second- ary peak at +5. The third example was M.BceJI, an MTase for which neither the target sequence, nor the type and position of methylation were known (Figure 5C). This MTase is part of a putative Type III restriction system from Burkholderia cenocepacia strain J2315 that is difﬁcult to transform. Type III REases rarely give complete digests making it difﬁcult to deduce their speciﬁcity. Since the speciﬁcity- deﬁning sequence is in the MTase protein, this means that the cognate MTases have previously been employed to determine speciﬁcity, but the approach needed is quite tedious (18). Kinetic signatures obtained from SMRT sequencing the plasmid expressing this Type III MTase were observed at every occurrence of the sequence context 50-CACAG-30 and at no other positions. For each occurrence, the second adenine position displayed kinetic signals of methylation to m6A (Supplementary Figure S4). Because the target sequence is non-palindromic and does not contain adenosine in the reverse complement, the methylation occurs only on one strand as is typical for Type III MTases (35). The presence of m6A in the 50-CACAG-30 sequence context was further validated by inhibition of digestion of an overlapping sequence by the methyl-sensitive ScaI restriction endo- nuclease (Supplementary Figure S5). Some MTases display off-target activities While the majority of MTases displayed kinetic signals present exclusively at their cognate sites indicating strict context speciﬁcities, we observed additional, off-target kinetic signals for some MTases. For example, a global analysis of M.EcoKDam modiﬁed DNA shows numerous PAGE 8 OF 12 smaller kinetic signals in addition to the 50-GATC-30 cognate site, suggesting partial methylation at off-target sites (Figure 4A, left panel). These off-target signals were found to encompass sequence contexts differing from the target context by one base (Figure 6). For M.EcoKDam, the 50-GACC-30 off-target context was most affected, followed by detectable signals for 50-HATC-30 (H = A, C or T) and 50-GATT-30. For example, the inset of Figure 6 shows IPD ratio data for the off-target 50-GACC-30 sequence: the lower average IPD ratio value is suggestive of partial methylation and in this case, methylation only occurs on one strand because there is no adenine in the reverse complementary sequence (50-GGTC-30). This example also exhibits a secondary IPD ratio peak at the +5 position which is commonly observed with m6A modiﬁcations in similar sequence contexts. The partial methylation of an off-target 50-GACC-30 site was conﬁrmed by showing 50% inhib- ition of SalI cleavage at an overlapping 50-GTCGACC-30 sequence (Supplementary Figure S6). DISCUSSION Thanks to the availability of a large number of cloned DNA methyltransferase genes that are expressed in an E. coli background devoid of other DNA methylation, it has been possible to prepare a series of pure plasmid DNAs each of which carries methylation signatures for just one methyltransferase. Using a few well-characterized methyltransferases as controls, we have been able to conﬁrm their methylation speciﬁcity using the SMRT sequencing approach and ﬁnd that in addition to obtain- ing interpretable signals for m6A and m5C as reported previously (28), it is also possible to detect m4C. This means that the three common types of methylation typic- ally found in bacterial and archaeal genomes can be un- ambiguously distinguished. Furthermore, when a suitable number of sites are present, both the type of methylation and a unique recognition sequence for the methyl- transferase can be assigned. By applying this same tech- nique to several methyltransferases with either unknown types and/or positions of methylation or in one case, both an unknown type of methylation and an unknown rec- ognition sequence, we have been able to assign speci- ﬁcity to the M.Tsp509I methyltransferase, the M.AatII methyltransferase and a new methyltransferase, M.BceJI, encoded by ORF 3494 in the B. cenocepacia genome. The latter is of particular interest because it is part of a Type III RM system and traditionally it has been quite difﬁcult to obtain the recognition sequences for these enzymes. The SMRT approach described here is well- suited for that purpose and if applied at a whole bacterial genome level, should also be suitable to obtain recognition sequences for Type I RM systems, which typically have been even more difﬁcult to determine than the Type III systems. Since most bacterial genomes contain many RM systems of all types, it seems likely that genome sequencing using the SMRT approach should, in many cases, allow the direct discovery of recognition sequences