Plasma and single-cell sequencing of multiple time points in a DLBCL patient (PT255). (A) Timeline of events for PT255. Clinical time point shows the timing of diagnosis (D) and relapses (R2, relapse 2; and R3, relapse 3) relative to blood sample collection (P1 to P6). Bulk tumor DNA was separately obtained from a biopsy at diagnosis, circulating tumor cells extracted at R2 and R3, and cfDNA extracted from plasma samples P1 to P6 after R2. Varying types of sequencing was performed on DNA from each time point, as summarized below. (B) Results from running PyClone on exome sequencing of DNA obtained from diagnosis, R2(P1,) and R3(P5). Clusters 0 and 1 contain trunk mutations seen at both P1 and P5; cluster 2 contains R2-specific mutations, and cluster 3 contains mutations that were subclonal at R2 and clonal at R3. (C) Amplicon sequencing of a subset of mutations found in the clusters in panel B from all 6 plasma time points reveal a more complete but similar evolution of the tumor as inferred from bulk sequence analysis in panel B. Below shows the suspected proportion of the tumor made up of each clone at individual time points. (D) Single-cell amplicon sequencing of circulating tumor cells taken at R2 and R3 revealed 2 distinct populations of cells containing mutations specific to each of R2 and R3. Genes are ordered by group and by frequency of mutation detected (top to bottom), suggesting a relative order of mutation acquisition.