Short DNA Probes Developed for Sample Tracking and Quality Assurance in Gene Panel Testing

Abstract

Both multiplexing and target-enrichment technologies are key to reducing the cost of genetic testing using next-generation sequencing (NGS). Many diagnostic laboratories routinely handle thousands of targeted resequencing samples, leading to an increased risk of accidental sample mix-ups and cross contamination. Herein, we present a short DNA fragment that can be spiked into the original genomic DNA (gDNA) or whole blood sample and tracked through to the final targeted resequencing data. This DNA fragment comprises a 15-bp unique index sequence assembled with a 120-bp fixed sequence designed for recovery in a hybridization capture reaction. In a pilot study, the yield of the recovered probe was examined in a step-by-step genetic testing procedure, involving gDNA isolation from whole blood, library preparation for NGS, and capture hybridization. On the basis of the results, 10 fmol (6×109 molecules) and 10 amol (6×106 molecules) of the spike-in probe were estimated to be suitable for DNA and RNA probe-based library preparation and target enrichment from 200 ng (6.5×104 copies) gDNA, respectively. In fact, the number of NGS reads corresponding to the spike-in probe was almost equal to that corresponding to the genomic target regions and was sufficient for evaluating sample identification and cross-contamination events. Hence, this method may be useful for enhancing quality assurance in clinical genetic testing.