Dynamic range, repeatability, and limits of detection and quantification

A dilution series in terms of target copies was prepared by mixing DNA from all 15 targeted transgenic soybean GM-events. Each GM-event was assigned with 128 target copies/µL, except for A2704-12 and MON89788, which had 49 and 92 copies/µL, respectively. The quantity of each soybean GM-event in the DNA mix was verified by duplex ddPCR (individual GM-event and Le1 in the same reaction; Supplementary Table ^S5). The verified values for the DNA mix were used to calculate the assigned copy numbers of the targets in a dilution series of the DNA mix (referred to as DNA mixes 1–10, with DNA mixes 2–10 representing 6×, 12×, 60×, 120×, 240×, 600×, 1200×, 6000× and 12000× dilutions of DNA mix 1, respectively). The final assigned values of the mixed DNA solutions were from 2527 to 0.02 copies of GM-event targets covered by the 6-plex, from 2761 to 0.05 copies of GM-event targets covered by the 7-plex, and from 4752 to 0.04 copies of GM-event targets covered by the 11-plex. The dilution series for all three multiplexes contained from 26789 to 0.07 copies of the endogene Le1. Twelve replicates of the dilution series were measured by ddPCR (three separate runs, in 3 days, each containing four replicates) for the 6-plex and 11-plex, and 14 replicates of the dilution series (three separate runs, in 3 days, with the first two days containing four replicates, and the last day containing six replicates) for the 7-plex. The absolute (copy number) limit of quantification (LOQ_abs) and detection (LOD_abs) for ddPCR were determined based on these experimental results. Another dilution series was prepared in terms of the GM-event target content, where DNA mix 1 was diluted in non-GM soybean DNA to obtain final concentrations from 0.005% to 2% of the GM-event target relative to Le1 (Supplementary Table ^S11). Three replicates of this dilution series were measured by ddPCR for each multiplex. The relative (%) limit of quantification (LOQ_rel) and detection (LOD_rel) for ddPCR were determined based on these experimental results. The LOQ (both absolute and relative) was determined as the lowest concentration in a series of concentrations that consistently yielded a relative standard deviation (RSD) <25%^{26, 27}. The LOD (both absolute and relative) was determined as the lowest concentration in a series of concentrations that consistently yielded positive signals with all replicates^{26, 27}.