PAGE 5 OF 12 Figure 2. Conﬁrmation of activity of cloned methyltransferase genes using methylation protection assays. Plasmids containing genes encoding the MTases (identiﬁed in the text above the gel images) were isolated from E. coli ER2796, a strain which lacks endogenous methyltransferase activities. Methylated plasmid DNAs (right lane in each case) were linearized using PstI then challenged with restriction endonucleases (REases) that are expected to be blocked by the action of the relevant cloned MTase. CviQI, RsaI, Sau3AI, Tsp509I, AatII, AluI, BstNI, NspI, HpaII and SacI samples were assayed using the equivalent cognate REases. EcoKDam and EsaLHCI samples were assayed using REase MboI, EsaBC1I using REase AluI, EcoKDcm Nucleic Acids Research, 2012, Vol. 40, No. 4 e29 simple means of visualizing the differences in polymerase kinetics between a native and control sample is to graph the ratio of IPDs (methylated/control) for each position along the DNA template. The extent and magnitude of these kinetic signals is dependent on the nature of the base modiﬁcation and the local sequence context. Because the polymerase is in contact with the modiﬁcation for several bases before and after occupying the polymer- ase active site, the kinetic signal can encompass multiple nucleotide incorporations surrounding the methylated position (28). We conﬁrmed the activities of the different MTases for all samples by restriction digestion (Figure 2). In all cases, the DNAs were protected completely from restriction digestion by the speciﬁc REase that corresponds to the particular MTase expressed on the plasmid, indicating complete methylation at the cognate sites (Figure 2). The validity of using WGA for generating the control data set for reference sequence and kinetics was conﬁrmed by analyzing a portion of the plasmid common to all samples, comparing the kinetics using as the control data either native, unampliﬁed DNA from the plasmid lacking an MTase gene, or whole-genome ampliﬁed (WGA) DNA derived from the MTase plasmid (Supplementary Figure S2). Characterization of MTase speciﬁcities We carried out SMRT sequencing on DNA templates with methylation marks from 16 different bacterial MTases, including enzymes with both known and unknown speciﬁcities (Table 1). Plasmid sequences were obtained through applying standard analysis tools (www .pacb.com/devnet), yielding high fold coverage over the entire DNA template from a single sequencing run (minimum of 300-fold; Supplementary Table S2), and are available at http://paciﬁcbiosciences.com/devnet/ﬁles/ how-tos/dna-methyltransferase/1.0/index.html. Consensus sequences of each of the plasmids can be found in Supplementary File S1. Results from the kinetic analysis are shown by example in Figure 3 for the two MTases, M.CviQI and M.RsaI, which modify the sequence context 50-GTAC-30 to impart m6A or m4C, respectively. For each MTase, the left panel shows a global analysis of the IPD ratio data for both strands of the entire plasmid. The right panels show detailed bar graphs of IPD ratios for two representative template positions. The large peaks of increased IPD ratio are present at sites of DNA modiﬁcation and correspond to the presumed recognition sequences for each bacterial DNA MTase, based on the known speciﬁcity of the cognate re- striction enzymes. As expected, the SMRT sequencing data from a plasmid lacking an MTase gene was devoid of any methylation-speciﬁc kinetic signals (Supplementary Figure 2. Continued with REase PspGI and EsaBC2I with REase SalI. Control PCR product DNAs (left lane in each case) were restricted with the same enzymes as the methylated plasmids but without PstI. The SacI un- modiﬁed (PCR product) DNA was also cleaved with PstI as this con- struct contains an additional PstI site within the coding sequence of the SacI MTase gene. M = 1 kb DNA-Ladder Marker.