An additional feature of the fragment identification procedure involves the modelling of potential hydrogen shifts. Transfer of one or more hydrogens during bond cleavages induced by UVPD is welldocumented in proteins and can also occur during photoactivation of nucleic acids. To capture these contributions to an isotopic distribution of a fragment ion, Nucleo-SAFARI employs a nonnegative least squares model to predict the relative amounts of the overlaid species arising from hydrogen shifts. A similar approach has been employed previously to perform relative quantitation of potential cytidine and uridine misincorporation in synthetic gRNAs. Identified fragment ions can be visualized, both in a sequence coverage map and interactive annotated spectrum, and with several built-in visualizations shown in Figures . These figures are generated within the application and display a summary of the total identified fragment abundances by type (Figure ), a summary of total identified fragment ion abundance by both type and cleavage position (Figure ), observed m/z error for all identified fragments (Figure ), and a summary of charge site localization based on the abundance of fragment ions in different charge states by cleavage position (Figure ). In addition to various visualizations, Nucleo-SAFARI also reports sequence coverage and a P-score. In brief, P-scores are calculated using a sequence tag approach, modified to account for the characteristics of nucleic acid fragmentation and consideration of fragment ions directly in the m/z domain. A more detailed description of the Pscore calculation is included in Supporting Information. Finally, .csv files containing the masses of the theoretical fragment ions (monoisotopic or isotopic distributions), fragment m/z values, and fragment identifications can be downloaded from within the application.