SPADA Draft Documents

number of iterations required to discover a high-performing multiplex reaction. No modeling 520 algorithm is perfect and there are many variables that are unknown even in the most 521 sophisticated design paradigm. However, using such sophisticated multiplex design will result in 522 many fewer design and experiment iterations and vastly superior assay performance.

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Metrology for In Silico Analysis

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Metrology is the science of measurement and serves an important, but often under- 526 appreciated role in the development and validation of in silico PCR assay design methods. 527 Measurement assurance concepts that help increase confidence and decrease uncertainty (the 528 error associated with a result) for experimental data (24) can also be applied to in silico 529 approaches.

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Sources of measurement uncertainty

One of the key steps in PCR assay design is predicting the outcome of applying an assay to 533 one or more DNA templates. While the information needed to define a PCR assay depends on 534 the complexity of the computational model, relevant information can include: 1) primer and 535 probe oligo sequences and concentrations; 2) template sequences and concentrations; 3) salt 536 concentrations; 4) thermocycling times and temperatures; 5) nucleotide concentrations; 5) 537 polymerase concentration and properties (i.e. nucleotide extension rate); and 6) buffer 538 composition. All of these parameters can affect the final outcome and therefore contribute to the 539 uncertainty, that is the error associated with the model prediction. 540 However, it is often challenging to obtain accurate, quantitative measurements for many of 541 these parameters in practice. Some information, like polymerase properties and the buffer 542

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