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

Multi-amplicon massively parallel sequencing confirmed 243 of 307 phenotype-filtered potentially pathogenic variants in 86 individuals with parkinsonism and controls, matching Sanger results.

• Why it matters: Whole-genome sequencing yields many variants, but only some need independent confirmation; Sanger is accurate yet too slow and costly for large-scale verification.
• What they did: The team built a custom amplicon panel with 290 amplicons targeting 307 variants, normalized and pooled DNA into 16 mixtures, sequenced with automated library prep, and assessed variant presence and quality computationally.
• The result: Unconfirmed targets included 18 sequencing artifacts, 5 insufficiently covered targets, 36 large indels >12 bp, and 5 homopolymer-region variants; concordance with Sanger and expected pooled frequencies support high-throughput confirmation, pending larger validation.

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