Multiplexed high resolution melting assay for versatile sample tracking in a diagnostic and research setting
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences . Biomedical Sciences
The journal of molecular diagnostics / American Society for Investigative Pathology; Association for Molecular Pathology [Bethesda, Md] - Bethesda, Md
, p. 32-38
University of Antwerp
Modern experimental procedures in molecular genetics, such as next-generation sequencing experiments, require that samples are taken along a whole series of wet- and dry-Laboratory steps. It generally is accepted that by increasing the complexity and number of steps in the experimental pipeline, the risk of sample swaps increases. It therefore is recommended to confirm the identity of each individual sample at the end of any pipeline. Here, we present a versatile assay to determine the identity of samples rapidly and efficiently by genotyping 21 single-nucleotide polymorphisms (SNPs) using multiplex high resolution melting. The selected SNPs also are present in whole-exome sequencing data, and comparison of the differentially obtained genotypes allows reliable identification of individual samples. In this assay, we combined primers interrogating two to three SNPs per high resolution melting reaction, enabling the generation of the SNP genotype profile in only eight reactions per sample, limiting the hands-on time and minimizing the amount of reagents. This SNP profiling approach also can be used to track samples in custom next-generation sequencing enrichment panels by including these 21 SNPs in the target region, allowing for the often-required independent validation of sample identity in both clinical and research settings.