Abstract Many mRNA vaccine production pipelines rely on Escherichia coli to replicate plasmid DNA templates used in the in vitro transcription of modified RNA. However, E. coli DNA methylation patterns differ substantially from those of humans. In E. coli, DNA methylation is primarily mediated by DNA adenine methyltransferase (Dam), which introduces N⁶-methyladenine (m6A) within GATC motifs, whereas human methylation occurs predominantly at cytosines in CpG dinucleotides. Some E. coli strains also express Dcm methyltransferase, which methylates CCWGG sequences (CC[A/T]GG), further distinguishing bacterial from mammalian epigenetic marks. Cytosolic DNA that lacks CpG methylation can potently activate Toll-like receptor 9 (TLR9), while m6A-modified DNA has been shown to stimulate the cGAS–STING pathway, leading to the induction of type I interferons and other inflammatory mediators. Because the Pfizer mRNA vaccine plasmids are propagated in E. coli, and residual plasmid DNA has been detected in finished vaccine material, it is likely that this DNA bears bacterial-type methylation patterns that could be immunostimulatory through TLR9 and cGAS–STING signaling. To investigate this possibility, we applied Oxford Nanopore sequencing to examine the methylation status of plasmid DNA present in Pfizer lot FL8095.