20.500.12556/RUL-178938 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. MtDNA next generation sequencing Sanger sequencing skeletal remains WWII sekvenciranje naslednje generacije Sangerjevo sekvenciranje skeletni ostanki druga svetovna vojna true false true Angleški jezik Slovenski jezik Članek v reviji 2026-02-02 12:42:06 2026-02-02 12:42:10 2026-02-03 04:26:08 0000-00-00 00:00:00 2026 0 0 Str. 169–174 iss. 1 Vol. 140 2026 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 61:340.6:902:94(100)"1939/1945" 1437-1596 10.1007/s00414-025-03603-1 251480579 s00414-025-03603-1.pdf s00414-025-03603-1.pdf 1 29D43DE53B40155D64E1672207EEF415 694939afe37fb752cdc90bf1cc24b7e5526bc327163a002b2a1dd93e5a10f10f bde4a8e5-002c-11f1-a1ba-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=226984 https://link.springer.com/article/10.1007/s00414-025-03603-1 1 24fb4628-002c-11f1-a1ba-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=226983 Medicinska fakulteta 0 0 0