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Simulate_PCR for amplicon prediction and annotation from multiplex, degenerate primers and probes
1Computations/Global Security, Lawrence Livermore National Laboratory (LLNL), Livermore, CA 94550, USA. gardner26@llnl.gov.
BMC Bioinformatics
|July 10, 2014
Summary
This study introduces Simulate_PCR, a command-line tool for predicting PCR product amplification from primer and probe sets against large sequence databases. It efficiently identifies all possible amplicons, accommodating primer mismatches and degenerate bases for high-throughput applications.
Area of Science:
- Bioinformatics
- Molecular Biology
- Computational Biology
Background:
- Designing multiplexed PCR assays requires careful primer selection to ensure target specificity and avoid cross-reactivity.
- Predicting primer and probe performance is crucial for successful PCR assay development, especially in high-throughput screening.
Purpose of the Study:
- To develop and present Simulate_PCR, a computational tool for predicting all possible PCR products from a given set of primers and probes against a large sequence database.
- To enable accurate prediction of amplicon sequences, including those with degenerate bases and primer-mismatches, and to identify probe binding sites.
Main Methods:
- The study describes a command-line Perl script that processes lists of primers and probes against a target sequence database (e.g., NCBI nt).
- The software identifies all potential primer-probe combinations, extracts amplicon sequences, and allows for user-defined mismatch tolerance.
- It calculates fragment length distributions for predicting gel electrophoresis patterns and annotates amplicons with gene information.
Main Results:
- Simulate_PCR successfully predicts and extracts all amplicon sequences from large databases, handling degenerate bases and primer mismatches.
- The tool indicates amplicons that can be detected by TaqMan®/Luminex® probes and annotates them with relevant gene information.
- It generates fragment length distributions to predict observable banding patterns on electrophoretic gels.
Conclusions:
- Simulate_PCR is a unique, freely available, open-source command-line tool for high-throughput PCR design.
- It simplifies the complex combinatorial challenge of primer-probe pairing without prior knowledge of pairings.
- The software has demonstrated utility in predicting PCR signatures for diverse targets, including viral genomes (HIV-1, Coronaviridae) and antibiotic resistance genes.
