title: Targeted resequencing and variant validation using pxlence PCR assays.
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.
authors.affiliation: Center for Medical Genetics, Ghent University, Ghent, Belgium; pxlence, Dendermonde, Belgium.
authors.forename: Frauke
authors.initials: F
authors.lastname: Coppieters
authors.affiliation: Center for Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Kimberly
authors.initials: K
authors.lastname: Verniers
authors.affiliation: Center for Medical Genetics, Ghent University, Ghent, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: De Leeneer
authors.affiliation: Center for Medical Genetics, Ghent University, Ghent, Belgium; pxlence, Dendermonde, Belgium.
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
authors.affiliation: Center for Medical Genetics, Ghent University, Ghent, Belgium; pxlence, Dendermonde, Belgium.
authors.forename: Steve
authors.initials: S
authors.lastname: Lefever
keywords: Amplification specificity; Next-generation sequencing; PCR; Sanger sequencing