Figure 1.

Uridine, ψ, and m1ψ are incorporated by T7 RNA polymerase with varying error rates during in vitro transcription. The RNA templates synthesized with T7 RNA polymerase were converted into cDNA with ProtoScript II reverse transcriptase followed by library preparation and sequencing. First-strand errors represented here stem from the combination of RNA polymerase and reverse transcriptase errors. (a) Combined first strand error rates are an average of four different RNA sequences (RNA1 and RNA2 are artificial RNA sequences that have been permutated to include every four-base combination; CLuc and BNT162b2 are functional mRNAs), and unpaired t-tests were used to calculate p-values. (b) Distribution of substitution, deletion, and insertion error percentages from four different RNA sequences. (c) Base substitution error profile observed for unmodified and modified RNA sequences are represented. Colors indicate specific substitution errors observed. Polymerase substitution errors are represented as the equivalent T7 RNA polymerase substitution (top substitution) or ProtoScript II reverse transcriptase substitution (bottom substitution).