e29 Nucleic Acids Research, 2012, Vol. 40, No. 4 PAGE 10 OF 12 Figure 6. Sequence context speciﬁcity of M.EcoKDam. Kinetic signals for the target 50-GATC-30 and all 1-base neighboring sequence contexts containing adenine at the second position are compared. The values plotted represent average IPD ratios of all occurrences of each sequence context (error bars are s.e.m.). The inset shows an example for a methy- lation signal at the off-target 50-GACC-30 sequence context. Error bars represent the standard error of the ratio of means and is calculated as described in the ‘Materials and Methods’ section. for the methyltransferases encoded by those genomes. Already, for all fully-sequenced genomes, REBASE (4) contains a theoretical analysis of the sequences and pre- dictions of the methyltransferase genes present in those genomes. In many cases, the recognition sequences of those methyltransferases can be inferred on the basis of sequence similarity and these predictions are included in the database. Conﬁrmation of many of these predictions should now be possible by SMRT sequencing, while in addition, the recognition sequences for many unassigned methyltransferases should also be obtainable. For all genomic sequences that become newly determined by the SMRT approach, the raw data will include both methyla- tion status, as well as just the raw sequence, a considerable bonus in terms of data acquisition. In previous studies of DNA methyltransferase speciﬁ- city, there has been no easy way to discover the ﬁdelity of the methyltransferase to ﬁnd out whether it matches the ﬁdelity of the cognate restriction enzyme. In the few cases where it has been possible to check this, the enzymes have shown the same degree of speciﬁcity of recognition as the restriction enzymes, but in at least one case, some prom- iscuity has been noted (36). It was thus of great interest to look at the promiscuity of the methyltransferases used in this study. For the Dam methyltransferase of E. coli (M.EcoKDam recognizing GATC) we found that, surpris- ingly, there was rather more off-target methylation than we anticipated. Given the role of the Dam methyltransferase in the initiation of DNA synthesis, one might have expected a high degree of ﬁdelity since this would seem to be a critical function and many GATC sites are found close to the origin. It appears that the initiation of DNA replication is not affected if additional methyl groups are present at non-cognate sites. Dam methyltransferase can affect transcription when GATC sites are methylated [reviewed earlier (5)]. The other known function of the Dam methyltransferase in damage repair would not be expected to be affected by off-target methylation. Whereas it is possible that the observed off-target signals are caused by mutation in the MTase genes present as a smaller fraction in the samples, we have not observed such minority species from SMRT sequencing, with a current detection limit of 2–3% (30). It should be noted though that for most of the methyltransferases that we studied, the ﬁdelity was actually quite high and relatively few off-target signals were found. This might be viewed as a little surprising since there is no clear biological selection mechanism that would ensure the accuracy of the methyltransferase component of an RM system. This is in contrast to the restriction enzyme itself where there is a strong selection for the cognate site since off-target cleavage known as star activity could easily cause damage to host genomes. In this context, it is worth noting that many restriction enzymes do show unwanted star activity (37) and it would be of considerable interest to check whether the methyl- transferases that accompany these promiscuous restriction enzymes show a similar promiscuity in their recognition, thereby protecting the organism against the potentially deleterious action of its restriction enzyme. We are greatly encouraged by the results of the studies described here, and plan to undertake a more systematic analysis of whole genome sequences, both with a view to examining the levels of DNA methylation in these genomes and possibly assigning speciﬁcity to the methyltransferases and correlating it with the predicted methyltransferase genes in the genome. Such studies will also provide the signiﬁcant quantities of data needed to enable exact quantitation of the degree of partial methy- lation and to deﬁne the precise sequences that lead to off-site methylation. By judicious analysis of methylation patterns, SMRT sequencing could also prove extremely useful in permitting the correct reassembly of individual genomes from complex metagenomic sequences. ACCESSION NUMBER Genbank accession number JN569339. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online: Supplementary Tables 1 and 2, Supplementary Figures 1–6 and Supplementary File 1. ACKNOWLEDGEMENTS We would like to thank L. Mazzola and N. Badger at the NEB sequencing core facility, D. Heiter, B. Jack, K. Lunnen, J. Samuelson, S.-Y. Xu and Z. Zhu for providing genomic or plasmid DNA samples containing MTase genes, and S.W. Turner, K. Luong, J. Bullard, J. Lee,