Natural CH in aged RMs. (A) Schematic workflow for error-corrected ultra-deep sequencing using a custom panel of primers covering RM homologs of 56 genes associated with human CH. Circulating granulocytes and dermal fibroblasts from aged RM (n = 60) were sequenced to detect somatic and germline mutations, respectively. (B) Cohort type and age distribution of the RMs enrolled in this analysis. (C) The number/percentage of aged RM carrying nonsynonymous driver CH somatic mutations identified by error-corrected deep sequencing (stratified by VAF ≥2%). (D) The number of driver CH mutations fitting conservative criteria in each of the 12 CH animals is depicted by age (red, VAF ≥2%; blue, VAF <2%). (E) The prevalence of aged RMs carrying CH somatic mutations stratified by age groups. Blue bars represent transplanted animals, and red bars represent nontransplanted naturally-aged animals. (F) Logistical regression analysis of age vs incidence of natural CH. Transplant recipients are excluded from this analysis. (G) The number and type of driver CH mutations are graphed by genes from the most to least frequent. (H) The VAF of each identified mutation are shown for each gene, from the most to least frequent. The bar represents the mean VAF of each gene, and the red dashed line indicates a 2% VAF. ATR, autologous transplant recipient; CR, calorie-restricted cohort; NM, nonmanipulated; OR, odds ratio.