Construction and validation of barcoded vector library. (A) Insertion of the barcode linker into retroviral vectors SF91 and MIEV. The linker contains a variable sequence part that consists of pairs of degenerate nucleotides (N or S) flanked by fixed triplets. The crude PCR sequence trace of the resulting vector batch suggests the random insertion of nucleotides at N and S positions. (B) The HC vector library, created by combining approximately 800 bacterial clones, was retransformed into Escherichia coli. By combining the sequence traces of the 88 resulting clones, the crude PCR sequence trace could be reconstructed. (C) Distribution plot showing observed and expected barcode frequencies for the HC vector library; given that 88 sequencing trials were performed. Binomial distribution modeling estimates a barcode complexity of approximately 440. (D) Distribution plot showing the observed number of nucleotide differences between all 78 barcodes in the HC library by performing pairwise comparisons. Also shown is the distribution of the predicted number of differences based on the simulation of 78 random barcodes (average of 10 simulations). LTR indicates long terminal repeat; Psi, packaging signal; eGFP, enhanced green fluorescent protein; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; pPGK, phosphoglycerate kinase promoter; IRES, internal ribosome entry site.