Detection of AID-induced mutations by NGS. NIH-3T3 cells were co-transduced with mOrange^STOP and AID-ER– or AID^E58Q-ER–expressing vectors. Transduced cells were cultured in the presence of OHT during 11 d. mOrange^STOP transgene was PCR amplified from the total cell population and mutations were analyzed by Sanger sequencing (A) and NGS (B and C). (A) Mutation frequency in AID-ER–expressing cells in the complete mOrange^STOP sequence (total) and in cytosines or guanines of AID hotspots (WRC/GYW and WRCY/RGYW) is represented. (B) Mutation frequency in AID-ER– or AID^E58Q-ER–expressing cells. Total mutation frequency was calculated as the mean of the mutation frequencies in all nucleotides of the amplified mOrange^STOP sequence. WRC/GYW and WRCY/RGYW mutation frequencies were calculated as the mean of the mutation frequencies in G/C nucleotides contained in the respective hotspots. Values are from one representative of three independent experiments (Table 2). (C) Mutation frequency in individual WRCY and RGYW hotspots in mOrange^STOP sequence, normalized to the mean mutation frequency at G/C nucleotides. Bars represent the mean of three independent experiments. Error bars show standard deviations. Shadowed hotspots (AACC, TGCT, GGTA, and GGTT) are not present in the mOrange^STOP sequence. The reference mOrange^STOP sequence is shown in Fig. S1 A.