The BREX engineered locus sequence was verified in two ways. First, PCR reactions (primers oJZ_241/242, S2 File ) with LongAmp polymerase (New England Biolabs; Ipswich, MA, USA), purified and sequenced (Sanger). Second, PacBio sequencing reads from the methylation analysis was aligned with the in silico design of the engineered locus.
DNA motifs and degree of modification were generated using InterPulse Duration (IPD) Ratios analyzed with RS_Modification_and_Motif_Analysis from Pacific Biosciences as in [93 (link),94 (link)]. PBMotStat, a component of REBASE TOOLS [95 (link)] was used to calculate the % of methylated sites with IPD > 2 for specific sites.
For the partially methylated strains, a.gff file of the methylation level of each base of the genome was downloaded from SMRT Link, filtered to keep only significantly methylated sites, and uploaded in Geneious Prime on the reference genome (S5 File ).
DNA motifs and degree of modification were generated using InterPulse Duration (IPD) Ratios analyzed with RS_Modification_and_Motif_Analysis from Pacific Biosciences as in [93 (link),94 (link)]. PBMotStat, a component of REBASE TOOLS [95 (link)] was used to calculate the % of methylated sites with IPD > 2 for specific sites.
For the partially methylated strains, a.gff file of the methylation level of each base of the genome was downloaded from SMRT Link, filtered to keep only significantly methylated sites, and uploaded in Geneious Prime on the reference genome (
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