Variant frequency distributions reflect pervasive subclonal representation of most somatic coding mutations in FL. (A) Kernel density plots (shaded gray) depict the distribution of somatic nucleotide variant frequencies in each of 8 patients. For each case, peaks can be seen corresponding to clusters of variants with different frequencies. For 6 of 8 cases, the major peak resides less than 20% frequency, indicating that the majority of mutations are present only in a minor subclone within the total tumor cell pool. Red vertical bars represent conservative estimates of tumor purity defined by IGH-BCL2 translocation qPCR assay (supplemental Figure 2). Vertical black bars in LPJ103 and LPJ108 represent homozygous CREBBP mutations that correlate with tumor purity. (B) Variant frequencies in 5 cases were corrected for tumor purity measured by IGH-BCL2 translocation and plotted against depth of sequencing for the given nucleotide. Two clear clusters can be seen, corresponding to mutations that are probably heterozygous in the entire tumor pool (uniformly heterozygous) or within a subclone (subclonally heterozygous). The remaining points probably represent mutations that are homozygous within the entire tumor pool (uniformly homozygous) or within a minor clone (subclonally homozygous). Notably, 3 of 4 MLL2 mutations (pink) fall within uniformly heterozygous cluster and the remaining mutation within the uniformly homozygous cluster. All 4 CREBBP mutations (blue) fall within the uniformly homozygous cluster.