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High-throughput PCR assay design for targeted resequencing using primerXL
Steve Lefever1,2,3,4, Filip Pattyn5,6, Bram De Wilde5,7,8
1Center for Medical Genetics, Ghent University, De Pintelaan 185, 9000, Ghent, Belgium. Steve.Lefever@UGent.be.
BMC Bioinformatics
|September 8, 2017
Summary
PrimerXL is a new webtool that designs PCR primers for targeted resequencing, overcoming limitations of current methods. It generates high-quality assays for cost-effective and efficient DNA sequencing projects.
Area of Science:
- Genomics and Bioinformatics
- Molecular Biology
Background:
- Whole genome sequencing is costly for routine use.
- Targeted resequencing reduces costs and data analysis complexity.
- PCR-based enrichment offers superior coverage uniformity, flexibility, and specificity compared to other methods, but requires extensive primer design.
Purpose of the Study:
- To develop an advanced PCR primer design pipeline for targeted resequencing.
- To overcome the limitations of current target enrichment strategies, particularly the extensive primer design needed for PCR-based enrichment.
Main Methods:
- Developed primerXL, a state-of-the-art PCR primer design pipeline.
- Utilized an optimized design criteria relaxation cascade.
- Implemented a thorough downstream in silico evaluation process.
- Generated high-quality singleplex PCR assays.
Main Results:
- PrimerXL outperforms other target enrichment strategies and primer design tools in assay quality, coverage uniformity, and target coverage.
- Demonstrated results from four sequencing projects involving 2348 amplicons covering 470 kb.
- Reduced the need for amplicon normalization.
Conclusions:
- PrimerXL is a user-friendly webtool for designing high-quality targeted resequencing assays.
- The customizable workflow and innovative relaxation cascade maximize target coverage.
- PrimerXL offers a cost-effective and efficient solution for targeted DNA sequencing.
