Computational tradeoffs in multiplex PCR assay design for SNP genotyping

Rachlin, John; Ding, Chunming; Cantor, Charles; Kasif, Simon
January 2005
BMC Genomics;2005, Vol. 6, p102
Academic Journal
Background: Multiplex PCR is a key technology for detecting infectious microorganisms, whole-genome sequencing, forensic analysis, and for enabling flexible yet low-cost genotyping. However, the design of a multiplex PCR assays requires the consideration of multiple competing objectives and physical constraints, and extensive computational analysis must be performed in order to identify the possible formation of primer-dimers that can negatively impact product yield. Results: This paper examines the computational design limits of multiplex PCR in the context of SNP genotyping and examines tradeoffs associated with several key design factors including multiplexing level (the number of primer pairs per tube), coverage (the % of SNP whose associated primers are actually assigned to one of several available tube), and tube-size uniformity. We also examine how design performance depends on the total number of available SNPs from which to choose, and primer stringency criterial. We show that finding high-multiplexing/high-coverage designs is subject to a computational phase transition, becoming dramatically more difficult when the probability of primer pair interaction exceeds a critical threshold. The precise location of this critical transition point depends on the number of available SNPs and the level of multiplexing required. We also demonstrate how coverage performance is impacted by the number of available snps, primer selection criteria, and target multiplexing levels. Conclusion: The presence of a phase transition suggests limits to scaling Multiplex PCR performance for high-throughput genomics applications. Achieving broad SNP coverage rapidly transitions from being very easy to very hard as the target multiplexing level (# of primer pairs per tube) increases. The onset of a phase transition can be "delayed" by having a larger pool of SNPs, or loosening primer selection constraints so as to increase the number of candidate primer pairs per SNP, though the latter may produce other adverse effects. The resulting design performance tradeoffs define a benchmark that can serve as the basis for comparing competing multiplex PCR design optimization algorithms and can also provide general rules-of-thumb to experimentalists seeking to understand the performance limits of standard multiplex PCR.


Related Articles

  • Morphologic and Genetic Identification of Taenia Tapeworms in Tanzania and DNA Genotyping of Taenia solium. Eom, Keeseon S.; Jong-Yil Chai; Tai-Soon Yong; Duk-Young Min; Han-Jong Rim; Charles Kihamia; Hyeong-Kyu Jeon // Korean Journal of Parasitology;Dec2011, Vol. 49 Issue 4, p399 

    Species identification of Taenia tapeworms was performed using morphologic observations and multiplex PCR and DNA sequencing of the mitochondrial cox1 gene. In 2008 and 2009, a total of 1,057 fecal samples were collected from residents of Kongwa district of Dodoma region, Tanzania, and examined...

  • WaferGen Biosystems' SmartChip to be tested at U. of Pittsburgh. Yoffee, Lynn // Medical Device Daily;11/16/2007, Vol. 11 Issue 221, p4 

    The article reports that the University of Pittsburgh Medical School will serve as the alpha test site for WaferGen Biosystems' SmartChip Real-Time Polymerase Chain Reaction (PCR) System. The SmartChip is designed as the first whole genome that delivers significant time and cost advantage to...

  • Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle.  // BMC Genomics;2011 Supplement 2, Vol. 12 Issue Suppl 2, p584 

    Background: Campylobacter jejuni and Campylobacter coli are human intestinal pathogens of global importance. Zoonotic transmission from livestock animals or animal-derived food is the likely cause for most of these infections. However, little is known about their general and host-specific...

  • Pathogen profiling for disease management and surveillance. Sintchenko, Vitali; Iredell, Jonathan R.; Gilbert, Gwendolyn L. // Nature Reviews Microbiology;Jun2007, Vol. 5 Issue 6, p464 

    The usefulness of rapid pathogen genotyping is widely recognized, but its effective interpretation and application requires integration into clinical and public health decision-making. How can pathogen genotyping data best be translated to inform disease management and surveillance? Pathogen...

  • CD14 C(-159)T Polymorphism Is a Risk Factor for Development of Pulmonary Tuberculosis. Rosas-Taraco, Adrian G.; Revol, Agnès; Salinas-Carmona, Mario C.; Rendon, Adrian; Caballero-Olin, Guillermo; Arce-Mendoza, Alma Y. // Journal of Infectious Diseases;12/1/2007, Vol. 196 Issue 11, p1698 

    Background. Neither the expression of CD14 and Toll-like receptor 4 (TLR4) on monocytes' surface nor the mutations CD14 -159TT and TLR4 Asp299Gly have yet been evaluated as risk factors for development of pulmonary tuberculosis (TB) in the Mexican population. Methods. Level of membrane CD14...

  • Genotipos de aislados de campo de Brucella abortus de distintas regiones geográficas de Chile. Mancilla, M.; Villarroel, M.; Saldías, M. E.; Soto, J.; Zárraga, A. M. // Archivos de Medicina Veterinaria;2008, Vol. 40 Issue 2, p187 

    Bovine brucellosis is an endemic, zoonotic disease of high economic impact. The genetic identification of the prevalent Brucella abortus strains, the pathogen, is key to pursue further epidemiological strategies for disease control. The insertion sequence IS711 has been used as genetic marker to...

  • Inferring ancient metabolism using ancestral core metabolic models of enterobacteria. Baumler, David J.; Bing Ma; Reed, Jennifer L.; Perna, Nicole T. // BMC Systems Biology;2013, Vol. 7 Issue 1, p1 

    Background: Enterobacteriaceae diversified from an ancestral lineage ~300-500 million years ago (mya) into a wide variety of free-living and host-associated lifestyles. Nutrient availability varies across niches, and evolution of metabolic networks likely played a key role in adaptation....

  • Analysis of the three STR loci (D16S539, D13S317, D13S317) in a population sample of Marmara region of Turkey. Çakir, A. Hadi; Şimşek, Fazilet; Çelebio&gcaron;lu, Ayşen; Taşdelen, Bahar // Journal of Cell & Molecular Biology;2002, Vol. 1 Issue 1, p25 

    In this study, allele and genotype frequencies for the short tandem repeat (STR) loci D16S539, D7S820, and D13S317 were investigated in 138 unrelated individuals (124 for D13S317, 125 for D7S820, 138 for D16S539) living in Marmara region by PCR technique using an allelic ladder consisting of...

  • PCR–RFLP based molecular typing of enteroviruses isolated from patients with aseptic meningitis in Korea. Lee, Y. S.; Kim, K. S.; Chung, Y. S.; Cheon, D. S.; Jung, S. K.; Park, K. S.; Na, B. K.; Jee, Y. M.; Kim, D. S.; Yoon, J. D.; Song, C. Y.; Lee, K. H. // Archives of Virology;Sep2002, Vol. 147 Issue 9, p1711 

    Summary. We have evaluated PCR–RFLP as a practical method for rapid typing of enteroviruses causing aseptic meningitis in Korea. Through blind examination of 80 clinical isolates from patients with aseptic meningitis, we have compared the results of conventional serotyping with...


Read the Article


Sorry, but this item is not currently available from your library.

Try another library?
Sign out of this library

Other Topics