Flowchart for generation of frequency distributions. Illustrated flowchart for the generation of several frequency distributions starting from a single experimental 96-well plate. Transgene (red) and reference gene (blue) are analysed via real-time PCR in separate wells. The light coloured wells contain reactions for 32 replicates of the sample DNA to be measured. Transgene and reference gene copy numbers are extrapolated from corresponding standard curves (dark colours). The white well depicts negative control. For each number of possible transgene or reference gene replicates (considered in two isolations each, i.e. 2× 2, 2× 3, 2× 4, 2× 5,…, 2× 16), copy numbers are arranged in 5000 combinations by chance. The resulting 5000 GMO percentages are divided into frequency classes and depicted. For each number of replicates, a corresponding frequency distribution with its statistical parameters can be visualised. Effects of different baseline/threshold settings are shown from left to right, increasing replicate numbers from top to bottom, respectively.