Barcode analysis of polyclonal and monoclonal 32D cell cultures. (A) Tracking the complexity of a polyclonal 32D cell culture over time. Subclone sequencing showed 77 unique barcodes of 105 sequencing trials after 2 weeks of culture, and 68 of 95 after 5 weeks of culture. White bars represent barcodes that were identified at 1 time point only, and colored/patterned bars represent barcodes that were found at both time points. (B) Distribution plots showing the observed and expected barcode frequencies for the 2 time points. Binomial distribution modeling predicts that the barcode complexity in this culture remained complex over time. (C) Crude PCR sequence traces of 2 monoclonal cultures provide an estimate of the total number of retroviral integrations per cell. The sequence trace for monoclone I shows that all variable positions in the barcode are restricted to 1 or 2 nucleotides, suggesting the presence of only 2 integration sites. The sequence trace for monoclone II is more complex, but 1 nucleotide is missing at several of the variable positions. (D) Subclone sequencing identified the unique barcodes that underlie the crude PCR sequence traces. Different barcodes are represented by different colors. Monoclone I contains 2 integrated retroviral vectors per cell, whereas monoclone II contains 9 integrated retroviral vectors per cell. Also shown are the actual barcode sequences identified for both monoclones. The sequence consensus pictograms show how the crude PCR sequence traces in panel B can be reconstructed by combining all identified barcodes in silico. (E) Gene expression analysis identified barcodes that were present at the RNA level. One of 9 barcodes in monoclone II (marked with an asterisk) appears to be overrepresented on the RNA level, but this finding does not reach statistical significance. Note that identical barcodes in panels D and E of this figure are represented with matching colors.