The --N7 option was added in v2.3 to allow processing and analysis of N7-G modifications induced under the experimental conditions specified for dms treatment - DMSmode. Traditionally, measurement and analysis of N7-G modifications on RNA has involved harsh biochemical processing separate from conventional N1/3 modification analysis. We have developed msDMS-MaP to allow simultaneous detection and analysis of both N1/3 and N7-G modifications. We have shown that N7-G reactivity is informative about RNA tertiary and quaternary structure. The N7-G reactivity analysis complements the traditional N1/3 reactivity analysis which is conventionally interpreted in the context of secondary structure.
Under the aformentioned experimental conditions, these N7-G modifications manifest as G>A mutations. Shapemapper isolates and processes these modifications in a channel separate from N1/3 modifications. When the --N7 flag is used, N7-G related information will be written to a profile.txtga file (and one or more .mutga files if the --output-parsed-mutations flag is used). Additionally, the N7-G data will be visualized alongside N1/3 data in the profile.pdf output file.
see DMSmode
Please see [place publication here] for a detailed description of N7-G normalization.
Due to the way these sites are normalized, the raw rate has as inverse correlation with normalized reactivity. In other words, an N7-G position with a low raw rate will have a high normalized reactivity. We term sites with high normalized reactivity "protected". Additionally, N7-G reactivity normalization incorporates a log2 transformation. Thus, successive increments of 1 correspond to a "doubling" of the N7-G normalized reactivity. For example a normalized reactivity of 2 is twice as protected as a normalized reactivity of 1 due to the preceding log2 transformation.
Based on prior experiments, we have set thresholds to determine how protected each N7-G position is. Cutoffs have been set at 1.6 and 2.3 corresponding to bases which are protected and highly protected respectively. In the profiles.pdf data visualization unprotected bases (N reactivity < 1.6) are colored black, protected bases (1.6 <= N reactivity < 2.3) are colored pink, and highly protected bases (N reactivity >= 2.3) are colored purple.
Additional analysis of N7-G data may be performed in RingMapper, DanceMapper, and ArcPlot.
Each of these packages has functionality specific to N7-G data processing.
Please cite Saleem and Miller et al, Journal To Be Determined, 202X, for publications using the --N7 option
Please cite Mitchell et al, Nucleic Acids Research, 2023, for publications using the --dms option
Bicine buffering conditions were first described in Mustoe et al, PNAS 2019