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4.3.11 Multiplexing is a complex system

Multiplex PCR is a complex system with many interacting variables (e.g., multiple primers, 525 genomic DNA from the target(s) of interest and contaminating genomes from the sample 526 matrix). Optimizing one set of primers can result in an unexpected interaction with another set of 527 primers in the reaction to create unexpected false amplicons from the exclusivity and background 528 databases or primer-amplicon cross-hybridization reactions. In addition, as the size of the 529 multiplexed reaction grows larger, there is a combinatorial explosion in the permutations of 530 possible multiplex reactions. Consider a 20-plex reaction with 10 primer design candidates (i.e. 531 the top scoring singleplex designs) for each of the 20 targets: the number of possible multiplex 532 reactions to consider is 10 20 . An iterative empirical approach can only assess a few reactions 533 (without knowledge of the interacting variables) and thus samples a very limited amount of the 534 sequence design space, almost always resulting in a sub-optimal design and often complete 535 failure. Even an iterative brute-force computational approach using high-performance computing 536 that could test millions of combinations of multiplex sets will vastly under sample the number of 537 possible reactions. For the 20-plex example with 10 candidate solutions, it would be 538 computationally intractable to run thermodynamic scanning (e.g., PrimerBLAST or 539 ThermoBLAST ) for all 10 20 possible multiplex permutations to ensure that false positives are 540 minimized. Thus, a modern sophisticated algorithm is needed to solve the combinatorial 541 explosion thoroughly and find the optimum multiplex design. The program PanelPlex uses a 542 depth-first search with a pruning algorithm to accomplish the multiplexing design task and 543 guarantees finding the optimum solution (or top N solutions) out of the entire space of possible 544 multiplexes. We recommend using such multiplex optimization algorithms to greatly reduce the 545

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