There is significant debate within the diagnostics community regarding the accuracy of variant identification by next-generation sequencing and the necessity of confirmatory testing of detected variants. Because the quality threshold to discriminate false positives depends on the workflow, no regulatory standard regarding this matter has yet been published. The goal of this study was to empirically determine the threshold to perform additional Sanger sequencing and to reduce the experimental cost to a practical level. Using 278 model genes, a hybridization capture-based protocol was examined to meet the clinical requirements of low cost, high efficiency, and high-quality data. To reduce excessive false-positive detection, filtering processes were introduced to remove mismapped reads and strand-biased detection to a published best-practices pipeline. With seven samples from the 1000 Genomes Project, 2750 single-nucleotide polymorphisms and 142 insertions/deletions were identified by our designed workflow. Compared with variants registered in the single nucleotide polymorphism database (dbSNP), a zero false-positive threshold value was determined (quality score > 1000). The variants satisfying these criteria accounted for 95.6% of single-nucleotide polymorphisms and 50.7% of insertions/deletions. Except for deletions located within the highly repeated sequences, the workflow achieved 100% sensitivity. The established threshold allowed us to discriminate between convincing variants and those requiring validation, a design that reconciles the competing objectives of cost minimization and quality maximization of clinical gene panel testing.

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 Â 10 9 molecules) and 10 amol (6 Â 10 6 molecules) of the spike-in probe were estimated to be suitable for DNA and RNA probeebased library preparation and target enrichment from 200 ng (6.5 Â 10 4 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.