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CREPE (CREate Primers and Evaluate): a computational tool for large-scale primer design and specificity analysis
Jonathan W Pitsch1, Sara A Wirth1, Nicole T Costantino1
1Department of Pediatrics, Section of Genetics and Metabolism, University of Colorado School of Medicine, Aurora, CO, USA.
Biorxiv : the Preprint Server for Biology
|April 16, 2025
Summary
CREPE is a new pipeline that automates DNA primer design and specificity checks, improving efficiency for large-scale projects. This tool enhances targeted amplicon sequencing by ensuring primer accuracy and reducing errors in biological research.
Area of Science:
- Molecular Biology
- Bioinformatics
- Genomics
Background:
- Polymerase chain reaction (PCR) is essential for DNA amplification but manual primer design is time-consuming and error-prone.
- Existing tools like Primer3 assist primer design, yet scaling and specificity assessment remain challenges for large datasets.
- Manual confirmation of primer specificity is crucial to avoid off-target amplification, adding complexity to high-throughput workflows.
Purpose of the Study:
- To develop an integrated computational pipeline, CREPE (Create Primers and Evaluate), for automated, large-scale primer design and specificity analysis.
- To fuse the capabilities of Primer3 and In-Silico PCR (ISPCR) to streamline primer selection and off-target evaluation.
- To provide a customized workflow for targeted amplicon sequencing (TAS) on Illumina platforms.
Main Methods:
- Integrated Primer3 and ISPCR functionalities into a novel pipeline named CREPE.
- Developed a custom evaluation script within CREPE for assessing primer specificity and likelihood of off-target binding.
- Implemented a workflow tailored for 150bp paired-end Illumina targeted amplicon sequencing.
Main Results:
- CREPE successfully performs primer design and specificity analysis for any number of target sites at scale.
- The pipeline outputs optimal primer pairs, off-target binding likelihood, and decision-support information for each target.
- Experimental validation demonstrated successful amplification for over 90% of primers identified as acceptable by CREPE.
Conclusions:
- CREPE offers a scalable and efficient solution for primer design and specificity assessment in large-scale genomics projects.
- The pipeline significantly reduces errors and time associated with manual primer design, enhancing experimental success rates.
- CREPE is a valuable tool for targeted amplicon sequencing, improving the reliability and throughput of DNA amplification strategies.
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