Figure 3.

Barcodes for tracking leukemia. (a) Schematic representation of vector ΔTrkA-LeGO-iG2-BC16 co-expressing the oncogene ΔTrkA and eGFP and equipped with a GFP-BC16 barcode library. The barcode library consisting of >700 000 different plasmids showed an equal distribution of the randomized nucleotides as evident from Illumina sequencing (>26 Mio reads) on 10¹⁰ plasmids (illustrated in the frequency plot). (b) Viral supernatant of the ΔTrkA-LeGO-iG2-BC16 plasmid library was used to transduce syngeneic lineage-negative bone marrow cells from male donors. Transduced cells were transplanted into lethally irradiated female recipient mice (n = 4). Control mice (n = 4) were transplanted with a barcoded eGFP marking vector (LeGO-G2-BC16). During follow-up, blood was taken every 4 weeks from transplanted mice. One mouse developed full-blown leukemia after 19 weeks as evidenced by the high proportion of eGFP-positive cells in the blood. All other mice showed stable eGFP counts in the peripheral blood during follow-up analysis. (c) Frequency analysis (stacked box plot) for barcodes found in leukemia samples by NGS of DNA from blood, spleen and bone marrow cells. The 10 most abundant barcodes were given individual colors, all other barcode sequences are summarized by gray boxes. (d) Sequences of the three leukemia-contributing barcodes, wobble bases are marked in accordance with the color of the respective box plot in (c).