Targeted resequencing and variant validation using pxlence PCR assays

Steve Lefever1,2, Frauke Coppieters1,2, Daisy Flamez3, Jo Vandesompele1,2
1Ghent University, Center for Medical Genetics Ghent, Ghent, Belgium; 
2pxlence, Dendermonde, Belgium; 
3Ghent University, Biomarked, Ghent, Belgium

Targeted resequencing has become an important application in clinical diagnostics. A wide range of target enrichment approaches are available, enabling the customer to focus on regions of interest. This not only reduces sequencing costs per sample but also facilitates downstream data analysis considerably. Due to its flexibility in design, high sensitivity and specificity, the polymerase chain reaction (PCR) is particularly well suited as enrichment strategy.
We developed and validated a primer design tool to generate one million PCR assays for both fresh frozen and formalin fixed paraffin embedded (FFPE) samples, covering over 99% of the human exome. Assays were designed to generate equimolar and specific amplification using uniform PCR conditions. Several proof-of-concept studies using the pxlence assays have been published. NGS gene panels were developed for congenital blindness (16 genes), deafness (15 genes) and various cancer types (16 genes). Uniform sequencing coverage has been achieved using different library preparations and sequencing instruments (GS FLX, Roche; GAII, MiSeq, Illumina). To date, the Center for Medical Genetics in Ghent uses the pxlence assays in a high-throughput singleplex enrichment workflow to replace Sanger sequencing-based diagnostic tests with NGS.
Due to the excellent performance of both the design pipeline and the generated primer pairs, a spin-off called pxlence (pronounced ‘pixellence’) was recently founded. Its short-term goal is to provide customers easy access to the predesigned assays, enabling them to enrich any exonic region or confirm any variant of interest through either NGS or Sanger sequencing. More information is available at

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