Supplemental Figure S5.

Representative example requiring additional merging in the final analysis. Not all sequences of the same size and family use can be merged as a single sequence. A: On the basis of the merged read summary output, there are two dominant sequences (rank, 1 to 2), but it is difficult to know where the background starts. The four top sequences (highlighted in green) show the same size but different VJ families. Sequences 1 and 2 are unrelated despite the same size and family (different subfamily), whereas sequences ranked 2, 3, 4, 7, and 8 are related and constitute the same clone with ongoing somatic hypermutation. B and C: Alignment of sequences 1 and 2 shows the sequences are widely different, whereas sequences 2 to 4 are related. D: Top panel: Snapshot of the V-J sequence frequency graph of LymphoTrack MiSeq Software version 2.3.1 shows a single bar with multiple colors (red and green are different subfamily). Sequences plotted by the Memorial Sloan Kettering LymphoClone viewer (middle) show sequences merged by VJ family use and size (single peak, two distinct colors). Bottom panel: Corresponding capillary electrophoresis tracing (FR1) also shows a dominant clonal peak and a small peak on the far right around 350 bp, which corresponds to the small sequence (rank, 5 and 6) 301 bp in length, V5-51_01;J6. In this case, the background is shifted down and begins at sequence rank 9 (highlighted orange). Corresponding flow cytometry identifies two distinct abnormal B-cell populations, 5.2% (λ restricted) and 7.9% (κ restricted) of the total white blood cells. N, no; Y, yes.