These open-source modules from the Cybertory platform are hosted here for use free of charge. Please contact us if you would like to install the programs and data sets on your own server. Using your own server may be more reliable for classroom use.
PCR is a central technique in molecular biology, with an extremely wide range of applications in genetic analysis and engineering. The PCR Simulator is a tool for teaching various aspects of this technology. It estimates the products of multiple primers acting on genome-scale templates under user-specified reaction conditions. Results are presented as images or animations of electrophoresis gels. A unique feature of this simulator is its support of individuals with different genotypes.
The PCR simulator takes user inputs for primer sequences and reaction conditions. It uses NIH BLAST to search the primers against genomic sequences to find the potential primer binding sites, then estimates the yield of each potential product using a quantitative model.
The Cybertory DNA Sequencing Simulator generates virtual DNA sequencing electropherograms based on the user’s choice of primer, template, and experimental parameters. Results are returned in Sequence Chromatogram Format (SCF), which can be read in third party trace viewers or sequence management programs, such as the Staden package.
Users choose a template and enter a primer sequence, and the sequencing simulator searches the template for potential primer binding sites. Each site is evaluated for priming efficency, using a nearest neighbor thermodynamic model. The program produces an SCF file containing the superimposed products from all priming sites, weighted by priming efficiency. This means that if the student has chosen a primer sequence that is not sufficiently unique, and/or has used insufficiently stringent hybridization conditions, the resulting sequence quality will be low, or perhaps unusable.
The sequencing simulator produces simulated results in a real data format (SCF). One of the major advantages of having the results in a real-world format like SCF is that they can be analyzed using real-world tools, like the Staden package. Traditionally, students have learned to use this software to assemble sets of sequence trace files. The advantage of having a sequencing simulator is that it closes the loop; finishing a sequence and resolving ambiguities is an iterative process. One often needs to perform additional reactions, often with custom primers, in order to obtain information necessary to join contigs, or to clarify uncertain sequence regions. This is not possible with static data sets.
The trace generator creates the chromatograms from the template sequence using its own model of the relationships between local sequence, primer position, peak height, and peak mobility. It then uses a "base caller" to essentially reverse the process, labeling the peaks to produce a sequence. By using a third-party base caller, with a slightly different model of the relationships between traces and sequences, we end up with occasional differences between the original template sequence and the simulated experimental results. This is a model of experimental uncertainty, and is a major component of our sequencing exercises.