Validation of the barcoding method in vitro and in vivo. (A) Experimental design. (B) Barcode composition in 1 representative cell culture; each color represents 1 barcode. (C) Quantification of barcode complexity in vitro. (D) Determinism in vitro barcode composition over time, calculated as Spearman rank coefficient between sequential passages of the same parallel culture. (E) In vivo human chimerism in xenografts of barcoded SupB15 leukemia cells, measured by flow cytometry for human CD19⁺ cells. One mouse did not engraft. (F-G) Barcode composition in blood at 5, 6, and 7 weeks after transplant and bone marrow (BM) of SupB15 xenografts. Each color represents 1 barcode; the height of the bars corresponds to the level of human chimerism. Depicted are 2 representative xenografts. (H) Comparison of barcodes in murine blood and in the in vitro culture over time. (I) Comparison of barcode clones in BM at the time of euthanization with in vitro clonality at the corresponding time points.