Pooled Multiplex Amplicon Massive Parallel Sequencing for High-Throughput Confirmation of Whole-Genome Sequencing Potentially Pathogenic Variants in Patients with Parkinsonism

Whole-genome sequencing identifies large numbers of genetic variants, although only a subset requires independent confirmation for clinical interpretation. Sanger sequencing remains the standard confirmation method but is impractical for large-scale variant verification. We developed and evaluated a strategy for variant confirmation based on multi-amplicon massively parallel sequencing of pooled DNA samples. Whole-genome sequencing data were obtained from 86 individuals, including patients with parkinsonism and controls. Variants were selected using phenotype-driven filtering, ClinVar classification, and pathogenicity assessment criteria. A total of 307 variants were targeted using a custom amplicon panel comprising 290 amplicons. DNA samples were normalized, pooled into 16 mixtures, and sequenced following automated library preparation. Variant presence and sequencing quality were assessed using standard bioinformatic tools. Of the 307 selected variants, 243 were successfully confirmed. Unconfirmed variants included sequencing artifacts (18), insufficiently covered targets (5), large insertions or deletions exceeding 12 base pairs (36), and variants located within homopolymeric regions (5). Results for selected variants were concordant with Sanger sequencing, and observed variant frequencies corresponded to expected frequencies in pooled samples. This approach provides a robust, rapid, and cost-effective method for high-throughput genetic variants confirming identified by whole-genome sequencing while requiring only limited DNA input. Despite complete concordance with Sanger sequencing, larger validation studies are needed before multi-amplicon massively parallel sequencing can replace routine confirmatory testing.