Protocol for streamlining genotyping of germline-transmissible mutants from genome-editing by using a parallel qPCR-based index and R analysis: GenoAlgo code

Quantitative analysis of the parallel qPCR results is performed using the GenoAlgo R program. The program imports the qPCR output file (Step 11) containing the Ct (cycle threshold) values obtained from reactions using the allele-specific primers (Rwt and Rm) paired with the common primer (Fq). For each sample, the program computes the relative signal values (Vwt and Vm) for the wild-type and mutant alleles, respectively. These values are normalized against the standard DNA dilution series generated in Step 5 to correct for amplification efficiency differences between primer sets. The iGenotype index is calculated using the formula: iGenotype = ((Vwt - Wm)/(Vwt+Vm))