e29 Nucleic Acids Research, 2012, Vol. 40, No. 4 recently been found to be a normal component of mam- malian DNA (12,13) and appears to be generated by oxi- dation of m5C in a reaction catalyzed by the ten eleven translocation (Tet)-family of enzymes (13). A number of diagnostic tools have been developed to detect DNA methylation, to establish what type of modi- ﬁcation is associated with a particular MTase and to de- termine the DNA sequence context in which such methylation occurs. Nucleosides containing m6A, m4C and m5C can be resolved from their unmodiﬁed equiva- lents by chromatographic methods, thereby facilitating their detection in total DNA hydrolysates (14,15). Alternatively, polyclonal antisera that speciﬁcally recog- nize m6A or m4C have been used for immunological detection of such modiﬁcations in order to ascribe function to multiple putative MTase genes in Helicobacter pylori (16). Bioinformatic analysis of a large number of genes that encode well-characterized prokaryotic MTases have identiﬁed groups of conserved sequence motifs that are diagnostic for DNA MTases and permit the accurate pre- diction of m5C-MTases, but the amino m6A- and m4C-MTases cannot be unequivocally distinguished (1,2). In the case of methylation activity that is part of a Type II RM system, the sequence speciﬁcity of the MTase is expected to be the same as the cleavage speciﬁcity of the associated REase. However, very little experimental evidence has been generated to support this, and biochem- ical characterization of the target speciﬁcity of most MTases remains to be gathered. Furthermore, the exact sites of modiﬁcation are often uncertain in cases where the recognition sequence contains multiple potential target bases (i.e. A or C) or where separate MTases act on the two strands independently (17). The experimental approaches currently available to fully characterize prokaryotic and eukaryotic MTases are labor-intensive, requiring radioactive labeling with 3 [ H] S-adenosylmethionine and mapping and sequencing of individual sites (17,18). One method based on Sanger sequencing can also detect methylated bases, but this is not a high-throughput method (19). Methods currently in use to discriminate between m5C and unmodiﬁed cyto- sines at CpG sequences, such as differential sensitivity to cleavage by REases (20) and methylation-speciﬁc polymerase chain reaction (PCR) of bisulﬁte-modiﬁed DNA (21,22) do not permit genome-wide analysis. The state-of-the-art method for m5C methylome studies is MethylC-Seq (23,24), but it is indirect and also requires extensive experimentation. In particular, the ﬁrst requisite is the complete DNA sequence of the DNA to be analyzed. Only then can methylome analysis be undertaken. Some additional new technologies have been described recently that have potential applications in methylome analysis such as methods to map genomic 5-hmC by enzyme-catalyzed glucosylation (25,26) and a family of REases that speciﬁcally recognize m5CpG and m5CpWpG sequences (27). The latter enzymes are of interest because members of this MspJI family of REases excise a 32–33 bp fragment that includes the methylated bases in a central position and the products lend themselves to high-throughput sequencing PAGE 2 OF 12 approaches. However, they are partially constrained by the sequence speciﬁcity of the REases (27). A method has been described previously to directly detect methylated DNA bases during single-molecule, real-time (SMRT) DNA sequencing (28). This method takes advantage of kinetic data pertaining to the rate of incorporation of each dNTP in the form of two param- eters—the pulse width (PW) and the interpulse duration (IPD). Signiﬁcant changes in these kinetic parameters were observed during SMRT sequencing when the DNA polymerase encounters m6A, m5C or 5-hmC on the template strand. These distinct kinetic signatures allow for the identiﬁcation of the type and position of the base modiﬁcation in the DNA template. Here, we extend the SMRT sequencing method to combine complete DNA sequence determination and methylated base analysis to characterize MTase speciﬁcities in a single operation (28,29). We analyzed a set of 16 DNA substrates that were methylated in vivo by a range of single prokaryotic MTases expressed in an E. coli strain that lacks additional MTase genes. The samples included MTases introducing m6A, m4C or m5C modiﬁ- cations, either from MTases whose substrate speciﬁcity was previously known, or from some whose speciﬁcity was unknown. The results allowed us to determine the absolute sequence speciﬁcity of the MTase, as well as the precise location of the methylated base. MATERIALS AND METHODS Materials All restriction endonucleases, Phusion-HF DNA polymer- ase, Antarctic Phosphatase, T4-DNA ligase, SAM, MTase genes and E. coli cells were from New England Biolabs Inc. (Ipswich, MA, USA). Synthetic oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). Escherichia coli strains C2523 (NEB-Express): fhuA2 [lon] ompT gal sulA11 R(mcr-73::miniTn10–TetS)2 [dcm] R(zgb-210::Tn10– TetS) endA1 (mcrC-mrr)114::IS10 C2925 (NEB dam-/dcm-): ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10) TetS endA1 rspL136 (StrR) dam13::Tn9 (CamR) xylA-5 mtl-1 thi-1 mcrB1 hsdR2 ER2796 (=DB24): fhuA2 D (lacZ)r1 glnV44 trp-31 dcm-6 his-1 zed-501::Tn10 argG6 rpsL104 dam-16::Kan xyl-7 mtl-2 metR1 mcr-62 D (mcrB-hsd-mrr)114 Methyltransferase cloning In total, 16 known and putative MTase genes (Table 1) were ampliﬁed from bacterial genomic or recombin- ant plasmid DNA sources with Phusion-HF DNA poly- merase using gene-speciﬁc oligonucleotide primers (Supplementary Table S1). The 50-end oligonucleotides incorporated a PstI site (SbfI site in the case of the gene encoding M.SacI which contains an internal PstI site), followed by the sequence 50-TTAAGG-30 (to terminate