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. 2010 Jun 23;11:340.
doi: 10.1186/1471-2105-11-340.

A High-Throughput Pipeline for the Design of Real-Time PCR Signatures

Free PMC article

A High-Throughput Pipeline for the Design of Real-Time PCR Signatures

Ravi Vijaya Satya et al. BMC Bioinformatics. .
Free PMC article


Background: Pathogen diagnostic assays based on polymerase chain reaction (PCR) technology provide high sensitivity and specificity. However, the design of these diagnostic assays is computationally intensive, requiring high-throughput methods to identify unique PCR signatures in the presence of an ever increasing availability of sequenced genomes.

Results: We present the Tool for PCR Signature Identification (TOPSI), a high-performance computing pipeline for the design of PCR-based pathogen diagnostic assays. The TOPSI pipeline efficiently designs PCR signatures common to multiple bacterial genomes by obtaining the shared regions through pairwise alignments between the input genomes. TOPSI successfully designed PCR signatures common to 18 Staphylococcus aureus genomes in less than 14 hours using 98 cores on a high-performance computing system.

Conclusions: TOPSI is a computationally efficient, fully integrated tool for high-throughput design of PCR signatures common to multiple bacterial genomes. TOPSI is freely available for download at


Figure 1
Figure 1
Components of a real-time PCR signature. A real-time PCR signature consists of a forward primer, a probe, and a reverse primer. The lengths of these three components, the distances between them, and the total amplicon length can vary greatly depending on the real-time PCR technology. The values shown in the figure are typical for the TaqMan® real-time PCR assays.
Figure 2
Figure 2
Overview of the TOPSI pipeline. The pre-processing stage of TOPSI obtains consensus sequence segments that are common to all input genomes. The actual signature design process, including comparison with non-target genomes, is performed in the three stages of the core TOPSI pipeline. The post-processing module assembles individual unique primers and probes into PCR signatures.
Figure 3
Figure 3
Distribution of TOPSI and KPATH signatures in the S. aureus Mu50 genome. The distributions shown here are for the 1236 KPATH signatures and the 2430 TOPSI signatures obtained using relaxed thresholds. Both TOPSI and KPATH signatures are distributed throughout the S. aureus genome. The regions in which there are very few or no TOPSI signatures also have very few or no KPATH signatures, indicating that these regions are in fact not suitable for PCR signature design.
Figure 4
Figure 4
Comparison of TOPSI signatures with experimentally verified signatures for B. mallei. The four TOPSI signatures and five experimentally verified signatures provided by the University of Maryland are mapped to the B. mallei ATCC 23344 genome.

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