Quantitative Characterization of Barcode Enrichment

gDNA was extracted from library-infected HPDE-iKRAS^G12D cells (injected or Input) and three individual tumour cores (output) for quadruplicate and triplicate, respectively, PCR reactions to amplify the BC pools present within each sample using Platinum Super Mix (Life Technologies) and flanking primers common to each BC: 5′-CAATTAACCCTCACTAAAGG-3′ and 5′-CCGCCACTGTGCTGGATA-3′). Amplification parameters were as follows: 1 × [94 °C-4']; 35 × [94 °C-1', 54 °C-1', 68-1']; 1 × [68 °C-10']. Each replicate PCR amplicon (178 nt) was purified for PGM library preparation, where each amplicon was ligated to unique Ion Xpress Barcode Adaptors for PGM sequencing (318 V2 Chip) following the manufacturer's recommendations. Raw data were concatenated into one ‘reference' file and indexed using burrows-wheeler alignment tool40 (link) for alignment of BC sequences (with parameters ‘-l7 -t12 -N -n3') for counting the occurrence of each BC. BC enrichment was assessed by quantitating the number of occurrences for each BC sequence as a ratio to total number of BC reads in each sample. S.d.'s were calculated for quadruplicate and triplicate reactions for input and output samples, respectively, and were plotted as error bars on the BC enrichment graphs.