FIG. 5.

Determination of relative frequencies for individual clones. (a) Calculation of clonal frequencies. The relative frequencies of VIS DNA of <500 bp from the left (x) and the right (y) junction were combined as described and represented as quantifiable vector integrants (QVIs). (b) The relation between the fraction of QVI in total vector (vertical axis) and expected overestimation (n-fold) of individual QVI frequency (horizontal axis). When 40.3% of vector integrants are QVIs, individual QVI frequencies are 2.56-fold overestimated. (c) Clonal frequency changes at four time points. The adjusted frequencies for individual QVIs were displayed according to the following color scheme: white to black to red, representing 0% to 0.1% to 3.1%. The frequency change of the top 15 highest-frequency QVIs was magnified on the right side. Ten of them were unambiguously mapped onto the rhesus genome (genomic locations are indicated on the right). Among those, seven were further tested with clone-specific real-time PCR. ND, nondetermined. (d) Clone-specific real-time PCR. The relative frequencies of seven QVIs were confirmed by clone-specific real-time PCR. Dark bars denote the adjusted relative frequencies determined by quantitative VIS sequencing. The values obtained by clone-specific real-time PCR (percentage of total vector copies) at the right junction and at the left junction of the vector integrants are shown with yellow and green bars, respectively.