Fig. 1: Development of CRACI for base-resolution quantitative sequencing of D.

a Schematic plot of the chemical principle underlying the reduction of D by KBH₄ treatment. b Comparative analysis of RT-truncation-based sequencing methods versus misincorporation-based methods for D detection. c Optimization of RT conditions increases misincorporation at D sites, shown by the misincorporation ratios of 256 NNDNN motifs. The violin plot (right) shows misincorporation distributions across 256 motifs under different conditions, with median (dashed line), interquartile range (dotted lines), data density (width), and extremes. Mutation patterns (T to C or T to A/G) are summarized as mean ± SD across 256 motifs. d Motif-dependent misincorporation patterns observed in CRACI using a synthetic 35-mer RNA oligo centered by NNDNN, in the presence of HIV RT and 1 mM/100 μM dGTP/dNTP combination. e Representative sequence-context-dependent calibration curves (misincorporation ratio vs. D fraction) for D stoichiometry quantification using CRACI. f A flowchart of CRACI library preparation, detecting D fractions through T-to-C misincorporation signatures.