Targeted resequencing and variant validation using pxlence PCR assays

Area of Science:

• Genomics
• Molecular Diagnostics
• Bioinformatics

Background:

• Next-generation sequencing (NGS) technologies have revolutionized molecular diagnostics.
• Polymerase chain reaction (PCR) is crucial for enriching target disease gene panels.
• A comprehensive primer design pipeline is essential for efficient exome coverage.

Purpose of the Study:

• To describe the assay specifications for a large set of PCR-based assays.
• To validate primerXL-designed assays using both in silico and wet-lab methods.
• To assess the performance of these assays in target enrichment for NGS and Sanger sequencing.

Main Methods:

• Development of a proprietary primer-design pipeline, primerXL, to generate over one million assays.
• In silico and wet-lab validation of 2294 selected assays.
• Utilized a universal PCR protocol without optimization for broad applicability.

Main Results:

• Assays cover over 98% of the human exome.
• Achieved high coverage uniformity and limited non-specific coverage with a universal protocol.
• Demonstrated a positive correlation between in silico specificity scores and actual assay performance.

Conclusions:

• The primerXL pipeline enables the design of robust PCR assays for comprehensive exome coverage.
• Validated assays exhibit high specificity and uniformity, suitable for molecular diagnostics.
• In silico prediction of specificity is a reliable indicator of assay performance.