SPADA Draft Documents

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Ranking the Design Results

A critical part of primer design is the ranking of the candidate designs using some sort of 433 scoring equation. Unfortunately, there is no agreed upon “currency” for goodness of primer 434 performance. Instead we have many metrics that have wildly different units, such as free-energy 435 differences for folding and hybridization, amount bound, amplicon folding, target conservation, 436 off-target hybridization, primer dimerization, primer-amplicon cross-hybridization, and a long 437 list of non-thermodynamic rules (G-quartets, sequence complexity (or information entropy), 438 amplicon length, etc.). It is still something of an art to combine all of these disparate scoring 439 terms into one big equation and to make it produce a result that is meaningful to a user (such as a 440 final score that ranges from 1 to 100). In addition, there is not agreement in the community as to 441 what the relative weighting of different scoring terms should be. For this reason, we recommend 442 using software that exposes the scoring equation and the weights used for each scoring term 443 (e.g., PanelPlex provides a detailed description of the scoring). Transparency by software 444 vendors regarding their scoring methods will give users the ability to change the scoring weights 445 and also to be more informed about what the modeling is and is not accounting for. In the future 446 when training and validation datasets become available as described in the Metrology section 447 below, the scoring terms and weights can be optimized by solving for the optimal weighting 448 terms. These datasets will also support the ability to evaluate the predictive quality of software 449 from different commercial and non-commercial sources.

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Combining All of the Recommendations into a Coherent Design Pipeline 452 In Figure 7, we provide an example of a design pipeline that combines the modern approaches 453 described in the above sections. Foremost in this modern approach is the integrated use of 454

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