Comparison of classic Sanger and next generation sequencing mitotypes of second world war victims from Konfin I mass grave.

Rapid technological advancements have significantly enhanced DNA analysis. A key innovation is Next-Generation Sequencing (NGS), also known as Massively Parallel Sequencing (MPS), which followed classic Sanger (CS) sequencing. Compared to CS, NGS offers higher sensitivity, resolution, and throughput, making it particularly valuable for mitochondrial DNA (mtDNA) analysis. The high copy number, matrilineal inheritance, and non-recombining nature of mtDNA, especially its hypervariable regions (HV), make it highly relevant in forensic investigations. NGS has introduced streamlined protocols and improved low-level heteroplasmy detection in mtDNA sequencing. However, with any new technology, its informativeness and authenticity must be evaluated against traditional methods. This study compared mitotypes from degraded WWII skeletal remains recovered from a Slovenian mass grave, using the same DNA extraction method to minimize pre-sequencing variability. Femurs were mechanically and chemically cleaned, pulverized, and fully demineralized. DNA was extracted and purified using EZ1 Advanced XL and quantified with an in-house protocol. CS sequencing was performed using BigDye Terminator Kit v1.1 and ABI PRISM™ 3130 Genetic Analyzer, while NGS was conducted with the Precision ID mtDNA Control Region Panel and Ion GeneStudio™ S5 System. Comparison of mitotypes revealed that NGS identified low-level heteroplasmies undetectable by CS, particularly in length heteroplasmy. However, since Ion Torrent™ Suite 5.10.1 is prone to errors, certain NGS variants had to be disregarded.