SPADA Draft Documents

composition, may be trade secrets that are not publicly shared (but some can be inferred or 543 measured). Other information, such as template concentration, are often measured in ways that 544 make biological sense (i.e. plaque forming units/ml, colony forming units/ml, etc.) but cannot 545 easily be converted into the units of Molarity required for modelling PCR with chemical kinetics 546 or equilibrium thermodynamics. In the absence of accurate input data, unknown model 547 parameters must be determined by fitting to measurements of PCR experiments. Note that 548 models are not restricted to “physical” parameters (e.g., reaction rates and concentrations), but 549 also include heuristic parameters such as sequence complexity and qualitative rules for primer 550 design, such as avoiding runs of guanines; providing bonuses for ending primers with “A” to 551 improve specificity; and similar patterns of sequences that should be avoided or enhanced even if 552 the detailed physical reasons are not known. Continued progress in machine learning raises the 553 possibility of training “black box” algorithms to predict PCR assay results based on a 554 combination of physical process models (e.g., DNA hybridization) and experimental assay 555 results. Regardless of how they are obtained, predicted PCR results should have an uncertainty 556 associated with them, in order to help interpret results and make decisions about assay 557 performance prior to moving to wet lab testing. 558 Another challenging aspect for metrology is defining the experimental quality of a given 559 PCR reaction. Two widely used experimental metrics are the “quantification cycle”, Cq, and the 560 PCR amplitude. The Cq is easy to determine from the experimental PCR curve (e.g., by 561 numerically determining the maximum second derivative point). For a given starting 562 concentration of target DNA, the Cq is a metric for the amplification efficiency of the PCR. 563 Primers that result in smaller Cq are generally considered to be superior to primers that result in 564 higher Cq. Reactions that have poor hybridization due to weak 2-state thermodynamics or 565

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