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composition, may be trade secrets that are not publicly shared (although some can be inferred or 560 measured). Other information, such as template concentration, are often measured in ways that 561 make biological sense (e.g., plaque forming units/ml, colony forming units/ml, etc.) but cannot 562 easily be converted into the units of Molarity required for modelling PCR with chemical kinetics 563 or equilibrium thermodynamics. In the absence of accurate input data, unknown model 564 parameters must be determined by fitting to measurements of PCR experiments. Note that 565 models are not restricted to “physical” parameters (e.g., reaction rates and concentrations), but 566 also include heuristic parameters such as sequence complexity and qualitative rules for primer 567 design, e.g., avoiding runs of guanines; providing bonuses for ending primers with “A” to 568 improve specificity; and similar patterns of sequences that should be avoided or enhanced even if 569 the detailed physical reasons are not known. Continued progress in machine learning raises the 570 possibility of training “black box” algorithms to predict PCR assay results based on a 571 combination of physical process models (e.g., DNA hybridization) and experimental assay 572 results. Regardless of how they are obtained, predicted PCR results should have an uncertainty 573 associated with them, in order to help interpret results and make decisions about assay 574 performance prior to moving to wet lab testing. 575 Another challenging aspect for metrology is defining the experimental quality of a given 576 PCR reaction. Two widely used experimental metrics are the “quantification cycle,” Cq, and the 577 PCR amplitude. The Cq can be determined from the experimental PCR curve (e.g., by 578 numerically determining the maximum second derivative point). For a given starting 579 concentration of target DNA, the Cq is a metric for the amplification efficiency of the PCR. 580 Primers that result in smaller Cq are generally considered to be superior to primers that result in 581 higher Cq. Reactions that have poor hybridization due to weak 2-state thermodynamics, 582

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