PCR based target enrichment for variant confirmation, gene panels and multiplex PCR sample tracking in a whole exome sequencing workflow

Background: Targeted PCR-based resequencing is an important application in clinical diagnostics. Using our best-in-class primer design tool primerXL, we have designed almost one million PCR assays for both fresh frozen and formalin-fixed paraffin-embedded samples, covering the entire human exome. Over 6200 assays for hundreds of clinically relevant genes in total were wet-lab validated. In addition, over 5000 patient-specific variants, from exome sequencing, were confirmed using pxlence PCR assays. All singleplex PCR assays work under universal PCR conditions and result in equimolar sequencing coverage. As a latest addition, we present the compatibility of pxlence assays with multiplex PCR applications. As a first product, we designed and validated a cost-effective and flexible sample tracking test. This primer pool enables fast identification of sample swapping or contamination which may occur in laborious library preparation workflows. Methods: Thirty SNPs were selected based on their minor allele frequency, exonic location and overlap with the capture region of exome enrichment kits. We evaluated three different mastermixes for multiplex PCR and two library preparation methods, followed by 150 bp paired-end sequencing on a MiSeq instrument (Illumina). Results: The SsoAdvanced PreAmp Supermix (Bio-Rad) resulted in superior homogenous coverage following multiplex PCR of all SNP assays (pxlence). No significant difference in coverage uniformity was observed between the Nextera DNA Flex and the NexteraXT DNA library prep method (both Illumina). In virtually all tested DNA samples (n=393), 86.29% of the SNPs had a uniform coverage within 2-fold of the mean. Based on the SNP genotypes, DNA samples could unambiguously be discriminated. Conclusion: In conclusion, pxlence provides high-quality and versatile PCR assays for various targeted resequencing applications. Here, we designed and validated a novel sample tracking test for whole exome or whole genome sequencing, involving a straightforward single multiplex PCR reaction followed by DNA sequencing library prep. In principle, our strategy could also be used to design gene panel-specific sample tracking solutions