High-throughput Amplicon Sequencing Optimization for Downstream Prediction Purposes

Abstract

Alongside short tandem repeat (STR) typing, forensic DNA phenotyping (FDP) can enhance forensic DNA analysis by inferring externally visible characteristics and biogeographical ancestry from single-nucleotide polymorphisms (SNPs). This project involves the optimization of a high-throughput amplicon sequencing assay targeting ~2,000 SNPs associated with pigmentation, facial morphology, and ancestry. Over 400 samples were processed through a genotyping pipeline involving DNA extraction, library preparation with a modified version of a commerical FDP assay, MiSeq FGx sequencing, genotype calling, and quality and ancestry inference assessments. The substantial dataset produced with this project will supply downstream applications in pigmentation and facial morphology prediction modeling. This work also advances FDP toward wide-scale integration into forensic laboratories and demonstrates how SNP-based genotyping can extend investigative leads when traditional STR matching is insufficient.