Of the two dominant methods today for target enrichment, multiplex PCR tends to have shorter workflows and require less DNA input than hybrid-capture probes 8. For routine detection of disease-relevant DNA variants in known genes of interest, targeted sequencing or direct qPCR approaches are typically used 6, 7. However, the human genome comprises over 3 billion nucleotides, and despite the lowering costs of high-throughput sequencing, it is not practical today to perform WGS to high depths necessary for identification of subclonal mutations, such as somatic mutations in cancer. For discovery applications, “shotgun” whole genome sequencing (WGS) is the preferred approach to identify novel DNA sequences of interest 5. The advance of high throughput sequencing has uncovered a large number of biomedically relevant DNA sequences, from driver mutations in cancer to new bacterial/viral pathogen DNA sequences to microbiome metagenomic profiles that affect mental disorders on the gut-brain axis 1, 2, 3, 4. In addition to NGS, SADDLE-designed primer sets can also be used in qPCR settings to allow highly multiplexed detection of gene fusions in cDNA, with a single-tube assay comprising 60 primers detecting 56 distinct gene fusions recurrently observed in lung cancer. Even when scaling to 384-plex (768 primers), the optimized primer set maintains low dimer fraction. In a 96-plex PCR primer set (192 primers), the fraction of primer dimers decreases from 90.7% in a naively designed primer set to 4.9% in our optimized primer set. Here, we present and experimentally validate Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE), a stochastic algorithm for design of multiplex PCR primer sets that minimize primer dimer formation. Simultaneously, there are exponentially many choices for multiplex primer sequence selection, resulting in systematic evaluation approaches being computationally intractable. One major challenge in the design of highly multiplexed PCR primer sets is the large number of potential primer dimer species that grows quadratically with the number of primers to be designed.
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