PrimerSuite

PrimerSuite designs PCR primers for bisulfite-converted DNA to enable locus-specific analysis of DNA methylation at CpG dinucleotides, including support for multiplex bisulfite PCR and downstream bisulfite resequencing.

Key Features:

• Bisulfite-aware primer design: Designs primers that accommodate bisulfite conversion chemistry by accounting for conversion of unmethylated cytosines to uracil and CpG methylation context.
• PrimerSuite module: Performs simultaneous design of bisulfite primers for multiple target regions to support parallel assay development.
• PrimerDimer module: Identifies and mitigates primer–primer dimerization artifacts to reduce non-specific interactions that compromise PCR specificity.
• PrimerPlex module: Supports design of multiplex PCR assays for concurrent amplification of multiple targets in a single reaction.
• Empirical validation metrics: Over 1,300 unique primer pairs were designed and screened, with >94% yielding amplicons of expected size and an average mapping efficiency of 93% by bisulfite multiplex resequencing.

Scientific Applications:

• DNA methylation analysis: Locus-specific interrogation of CpG methylation using bisulfite PCR.
• High-throughput bisulfite workflows: Design and validation of primers for bisulfite multiplex PCR and downstream bisulfite multiplex resequencing.
• Epigenetics research: Development of targeted assays for studies of methylation-mediated regulation.
• Related bisulfite methods: Potential application to bisulfite-based assays such as methylation-specific PCR.

Methodology:

Computational workflow comprises three modules that perform simultaneous bisulfite primer design for multiple targets, in silico identification and mitigation of primer–primer dimers, and multiplex PCR assay design.