Fig. 5: Characterization of functional ALK fusions identified in osimertinib-resistant PC-9 cells by HTGTS.

a The total number of translocations in before and after osimertinib selection by HTGTS. n = 3 HTGTS experiment was performed and pooled for DNA breakpoint analysis. b, c Rainfall plots showing genome-wide distribution of translocations before (b) and after (c) osimertinib selection in PC-9 cells. Each dot represents a single DNA translocation ordered on the X-axis according to its position in the human genome. Red and blue dots represent the orientation of DNA translocations on chromosome plus or minus strand, respectively. Example genes that transcribe in the plus and minus directions are shown in blue and red, respectively, resulting in functional ALK fusions in the correct direction. Data pooled from six biological replicates. d Venn diagram showing the overlap of shared DNA translocation hotspots between before and after selection. CRISPR off-targets were excluded. e, f Change in the number of DNA translocations in 154 hotspots before (e) and 16 hotspots after (f) osimertinib selection. g Distribution of DNA breakpoints and histone modification marks in RPL3 gene. Transcription start site (TSS) is indicated in pink. h Frequency of ALK translocations with the indicated partner genes. Each color corresponds to each gene. i Box plot showing the strand bias score of DNA translocations in 154 hotspots before and 16 hotspots after osimertinib selection. Y-axis represents strand bias score where 1 indicates no strand bias and 0 a 100% strand biased. The minima and maxima of box indicate the 25th to 75th percentile. The centre indicates 50th percentile. The wiskers were drawn down to the 10th percentile and up to 90th percentile. Significance was determined by an unpaired, two-tailed Student’s t-test. j Pie graphs showing translocation distributions analyzed from hotspots identified before (left) and after (right) osimertinib selection. k Detailed distribution of DNA breakpoints in EML4 genes. The purple arrows indicate orientation of genes. The number of translocations in focal clusters is indicated in black and red for before and after selection, respectively. Source data are provided as a Source Data file.