Development and Validation of PCR-Based Ancient DNA Analysis for Italian and Western Balkan Archaeological Collections.

Introduction: The Italian Peninsula and Western Balkans preserve rich archaeological records that extend from pre-Roman times through the Greek colonial, Roman, and Byzantine periods. Genetic investigation of these collections often requires access to specialized high-throughput technologies which are not commonly available or cost-effective method to handle the problem of broken skeletal remains. Methods: We developed and validated an ancient DNA protocol for Italian and Albanian archeological skeletal samples spanning approximately 2500 years. The protocol included artificial fragmentation of modern DNA to simulate ancient molecular profiles and validation using archaeological remains from Sicily, Rome and Apollonia. The workflow integrated EDTA demineralization, silica-based purification, PCR targeting short amplicons (<150 bp) to enable sex determination, species authentication, uniparental haplogroup analysis, and pigmentation trait prediction. Strict anti-contamination and authentication criteria were enforced. Results: Amplifiable DNA was recovered from all tested skeletal samples. Mitochondrial DNA haplogroups were assigned in 82%, while Y-chromosome markers were found in 76% of males, while sex was determined in 85% of the samples. Pigmentation SNPs yielded complete profiles in 78%. Amplicons <110 bp showed >90% amplification success. All results were reproducible and met authentication criteria, including phylogenetic plausibility and inverse fragment length-yield correlation. Conclusion: This protocol enables ancient DNA analysis from degraded skeletal remains of Mediterranean archaeological contexts. The simulated ancient DNA presents a key innovation, allowing refinement of procedures before application to irreplaceable material and provides a practical approach for laboratories with limited access to High-Throughput sequencing. The research is a practical framework for studying mobility, ancestry and demography among ancient populations from the Mediterranean and Western Balkan regions.