Targeted resequencing and variant validation using pxlence PCR assays

Frauke Coppieters; Kimberly Verniers; Kim De Leeneer; Jo Vandesompele; Steve Lefever

doi:10.1016/j.bdq.2015.09.001

Targeted resequencing and variant validation using pxlence PCR assays

Biomol Detect Quantif. 2015 Oct 9:6:22-6. doi: 10.1016/j.bdq.2015.09.001. eCollection 2016 Jan.

Authors

Frauke Coppieters¹, Kimberly Verniers², Kim De Leeneer², Jo Vandesompele¹, Steve Lefever¹

Affiliations

¹ Center for Medical Genetics, Ghent University, Ghent, Belgium; pxlence, Dendermonde, Belgium.
² Center for Medical Genetics, Ghent University, Ghent, Belgium.

Abstract

The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

Keywords: Amplification specificity; Next-generation sequencing; PCR; Sanger sequencing.