Optimization and Validation of Multimodular, Long-Range PCR-Based Next-Generation Sequencing Assays for Comprehensive...

The genetic diagnosis of tuberous sclerosis complex is difficult because of its broad spectrum of mutations. In addition to point mutations in coding regions, intragenic or chromosomal-level large deletions, deep intronic splicing mutations, and mosaic mutations represent a significant proportion of the mutations. In this study, multimodular, long-range PCR-based next-generation sequencing assays were optimized and validated using >100 samples with known TSC1 and TSC2 variants. Multiplex, long-range PCR covering the entire genomic region of both genes detected all 138 known variants; however, it also yielded false-positive results. Intragenic large deletions were detected with accurate breakpoint sequences. Chromosomal-level deletions were estimated by discordant allele segregation in the family and confirmed by DNA microarray. Deep intronic mutations were verified using a combination of long-range DNA PCR and full-length mRNA sequencing. DNA samples were mixed to simulate mosaic mutations, and most variants were detected but could not be distinguished from equivalently detected false-positive results. Repeated false-positive results were classified, and the strategy of selecting the common variants detected in the duplicate analysis and eliminating known false-positive results improved the sensitivity (85.2%) and positive predictive value (96.6%) of a 10% mosaic simulation. Long-range PCRbased next-generation sequencing is a highly versatile genetic test; however, confirmation tests remain necessary for clinical use because false-positive results cannot be completely eliminated from single experiments.