Data analysis using Nucleo-SAFARI involves three major steps, illustrated in Second, represented in Figure , the net chemical formula of the intact nucleic acid and its fragment ions are determined as a total sum of each element for the intact precursor, or as a rolling cumulative sum from the 5' or 3' direction for fragment ions. Monoisotopic masses of each fragment ion are calculated directly from the generated chemical formulas. Fine isotopic structures are generated up to a total probability of 97% using the IsoSpecR package, and the probabilities of each A more detailed version of the fragment identification procedure is outlined in Figure . Figure displays a well-resolved fragment ion in the m/z domain, which is identified against theoretical m/z values of various fragment ions within a 10 ppm m/z tolerance in Figure . Four theoretical fragment ions are displayed as patterns of short color-coded bars in Figure . From resulting m/z identifications, predicted abundances are determined for each isotope based on the probabilities of the theoretical isotopic distribution and total abundance of the observed isotopic distribution in Figure . At the same time, the first validation steps take place, where the identified isotopes must be sequential, and the total theoretical probability of these identified theoretical isotopes must exceed 70%, encompassing a majority of the predicted isotopic abundance. In practice, this step mitigates false identification of fragment ions that would be removed during validation as well as false identifications of isotopic distributions with half the charge and mass of the theoretical fragment. Last, in Figure , the observed isotopic distribution is validated against the predicted isotopic abundances based on a weighted average error between the observed and predicted abundance of each isotope (weighted by isotope probability). If this average error is less than a userdefined tolerance (default 25%), the fragment ion is considered a match, as is the case with the w12 ion of a 60 mer DNA strand in Figure .