Variant sequence patterns allow identification of 10/10 FL tumor populations from pooled
reads. (A) Reference sequence for BCL2 ROA 447 to 510 and dictionary with associated frequencies (word occurrence/total coverage) are shown for the 12× blended BCL2 analysis. Red letters in the reference sequence show 15 locations with identified base changes (a-o). The dictionary of verified words indicates reference identity with dots and changed bases with letters. Circled bases represent validated SNV calls not reported by VarScan2 due to presence of higher frequency variants occurring at identical locations. An inferred evolutionary interpretation of each word, based on phylogenetic analysis by maximum likelihood method (see 9C), precedes the word sequence, identifying 10 out of 10 known subpopulations within the mixture at this location, with three subpopulations showing additional mutations (clones 2, 3, and 10). (B) VarScan2 BCL2 results at 0.5% threshold for locations covered by BCL2 ROA 447 to 510, indicating reference location (a-o), associated base change and frequencies in the range of 1.06% to 6.60%. Note how ancestral or coincidental mutations are aggregated in the frequency determination, confounding the use of similar frequencies to identify subclonal specific mutation patterns. (C) This dendrogram was generated from DDiMAP dictionary: BCL2 ROA 477 to 510 using MEGA6 , a freely-available online tool for evaluating evolutionary relationships based on sequence analysis. The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura-Nei model . The tree with the highest log likelihood (-142.0600) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.