Fig. 5: Analysis of reference eGFP mRNA vaccine incorporating modified nucleosides using direct RNA sequencing.

a, b Genome-browser (IGV) view of long-read (ONT) alignment to the plasmid reference, sequenced using direct RNA sequencing for mRNA vaccines prepared with a Uridine and b N1 methylpseudouridine. Coverage indicates the number of reads at each nucleoside position while the lower alignments grey bars indicate unique, individual alignments, with colouring indicating their similarity to the reference genome. Heterogenous coverage is observed in the direct RNA sequencing, likely due to fragmentation of the modified mRNA vaccine. c mRNA length analyses demonstrate shorter length for modified mRNA vaccines due to fragmentation and enrichment in deletion sequencing errors (n = 4). d Direct RNA sequencing shows stereotypical errors (cytosine, blue; uridine; red) at N1-methylpseudouridine compared to uridine nucleosides. e Cumulative distribution plot shows per-nucleoside error profile for N1-methylpseudouridine (red/orange) compared to unmodified nucleosides (blue/green). Source data are provided as a Source data file.