Methods and related aspects for quantitative ratiometric regression polymerase chain reaction

A method of generating a standard curve for determining a fractional abundance of at least one target variant in a population at least partially using a computer, the method comprising:

• amplifying at least a first target nucleic acid and at least a first reference nucleic acid together in a reaction mixture to produce amplification data that comprises one or more detectable signal levels detected as the first target nucleic acid and the first reference nucleic acid are amplified, wherein at least initial fractional abundances of the first target nucleic acid and the first reference nucleic acid in the reaction mixture are known;
• subtracting, by the computer, at least one baseline detectable signal level value from the amplification data to produce baseline adjusted data;
• determining, by the computer, one or more ratios from the baseline adjusted data at one or more amplification cycles to produce at least a first set of amplification ratios;
• calculating, by the computer, adjusted cycle numbers for the first set of amplification ratios to produce at least a first set of adjusted amplification data;
• fitting, by the computer, fractional abundance values to the first set of adjusted amplification data using at least one fitting technique to produce fractional abundance fits;
• and,
• selecting, by the computer, a best fit for modeling fractional abundance from the fractional abundance fits, thereby generating the standard curve for determining the fractional abundance of the target variant in the population.

A system, comprising a controller comprising, or capable of accessing, computer readable media comprising non-transitory computer-executable instructions which, when executed by at least one electronic processor perform at least:

• determining a ratio of detectable signal levels detected for at least a first target nucleic acid to detectable signal levels detected for at least a first reference nucleic acid to produce at least a first set of relative detectable signal level ratios from amplification data that comprises one or more detectable signal levels detected as the first target nucleic acid and the first reference nucleic acid are amplified in a reaction mixture;
• determining a ratio of the detectable signal levels detected for the first target nucleic acid to a cumulative total of the detectable signal levels detected for the first target nucleic acid and for the first reference nucleic acid to produce at least a first set of cumulative detectable signal level ratios;
• and,
• determining a fractional abundance of at least a first target variant in a population at and/or after the quantitation cycle (Cq) value for the first target nucleic acid from the first set of relative detectable signal level ratios and/or the first set of cumulative detectable signal level ratios and at least one N-dimensional standard curve generated from calibration data of known fractional abundance, where Nis a number of ratios at different cycle numbers, to determine the fractional abundance of the target variant in the population.