No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 M.CviQI M.RsaI M.EcoKDam M.EsaLHCI M.Sau3AI M.AluI M.EsaBC1I M.BstNI M.EcoKDcm M.EsaBC2I M.NspI M.HpaII M.SacI M.Tsp509I M.AatII M.BceJI GT(m6A)C GTA(m4C) G(m6A)TC GAT(m4C) GAT(m5C) AG(m5C)T AG(m4C)T C(m4C)WGG C(m5C)WGG T(m4C)GA R(m5C)ATGY C(m5C)GG GAG(m5C)TC AATT (m6A; position unknown) GACGTC (m6A/m4C; position unknown) Unknown 3566 3986 3592 3590 3995 4331 3797 4142 4175 3566 3950 3833 3936 3863 3750 4760 3 2 23 19 19 19 18 5 7 2 3 15 1 7 2 Unknown PAGE 3 OF 12 Nucleic Acids Research, 2012, Vol. 40, No. 4 e29 Table 1. MTases subjected to speciﬁcity characterization by SMRT DNA sequencing Methyltransferase name Methylation context Plasmid size (bp) No. of sites translation of the lac a-peptide reading frame of the pRRS plasmid vector and reinitiate translation of the cloned MTase genes), followed by an eight nucleotide spacer sequence 50-TTAATCAT-30 and sequences complemen- tary to the 50-end of the relevant MTase coding sequence. 30-end oligonucleotides were complementary to the 30-end of the MTase coding sequences, including trans- lation termination codons and either BamHI or BglII re- striction sites. Because the predicted sequences of the pRRS constructs containing the M.EsaBC2I and M.SacI genes do not contain any restriction sites that are diagnos- tic of the activity of these MTases, additional restriction site sequences (for SalI and SacI, respectively) were included in the 30-oligonucleotides, positioned between the termination codon and BamHI site in each case. PCR amplicons were restricted with PstI (or SbfI) and BamHI (or BglII) and ligated to PstI–BamHI restricted pRRS plasmid DNA (Genbank accession number JN569339) that had been dephosphorylated using Antarctic Phosphatase. All methylase genes were under the control of the same E. coli promoter present in the pRRS vector. Isolation of plasmid DNA and determination of methylation status Ligation products were used to transform NEB-Express E. coli (or NEB dam-/dcm- E. coli in the case of the M.Sau3AI ligation) and recombinant plasmid DNAs were isolated from ampicillin-resistant transformants and the presence of inserts of the expected size was con- ﬁrmed by restriction analysis. Plasmids were then used to transform ER2796, also called DB24, a strain that lacks all known E. coli MTase genes (16). Plasmid DNAs were reisolated from ER2796 cells and their methylation status was assessed by restriction with PstI plus the relevant cognate restriction endonuclease in the cases of constructs containing M.AatII, M.AluI, M.BstNI, M.CviQI, M.HpaII, M.NspI, M.RsaI, M.SacI, M.Sau3AI and M.Tsp509I. Constructs containing genes encoding M.EcoKDam, M.EcoKDcm, M.EsaBC1I, M.EsaBC2I and M.EsaLHCI were assessed by restriction with PstI plus MboI, PspGI, AluI, SalI and MboI endonucleases, respectively. Unmethylated control substrates for each construct were produced by PCR ampliﬁcation (using Phusion-HF polymerase) of the complete plasmids— using oligonucleotide primers that anneal to opposite strands of the vector DNA at a position 18 nucleotides 50 of the vector SbfI/PstI site. Control substrates were re- stricted with the same enzymes as the methylated plasmids but without PstI (except in the case of M.SacI due to the internal PstI site within the gene). Sample preparation and SMRT sequencing An aliquot of 25 ng of plasmid DNA was whole-genome ampliﬁed (WGA) using the REPLI-g Midi Kit (Qiagen, Valencia, CA, USA). Ampliﬁcation factors were typically approximately 1600 ( 40 ug output), translating to <0.06% of residual methylated DNA in the control samples. Further trimming of 5% of data from the top and bottom of the IPD distributions (see data analysis section) removed any remaining spurious signal from modiﬁed DNA. WGA and native plasmid DNA was sheared to an average size of 300 bp via adaptive focused acoustics (Covaris, Woburn, MA, USA). SMRTbell template sequencing libraries were prepared as previously described (30). Brieﬂy, sheared DNA was end repaired, A-tailed and hairpin adapters with a single T-overhang were ligated. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA). Primer was annealed and samples were sequenced on the PacBio RS as previously described (31,32). Data processing Reads were processed and mapped to the respective refer- ence sequences for each plasmid using the Basic Local Alignment with Successive Reﬁnement (BLASR) mapper (http://www.pacbiodevnet.com/smrtanalysis/software/ blasr) and the Paciﬁc Biosciences SMRT Analysis pipeline (http://www.pacbiodevnet.com/smrtanalysis/software/ smrtpipe) using the standard mapping protocol. Interpulse