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

Obal, Marcel (author), Zupanič-Pajnič, Irena (author)

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.

• Language: English
• Keywords: MtDNA, next generation sequencing, Sanger sequencing, skeletal remains, WWII
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2026
• Number of pages: Str. 169–174
• Numbering: Vol. 140, iss. 1
• PID: 20.500.12556/RUL-178938
• UDC: 61:340.6:902:94(100)"1939/1945"
• ISSN on article: 1437-1596
• DOI: 10.1007/s00414-025-03603-1
• COBISS.SI-ID: 251480579
• Publication date in RUL: 02.02.2026

Record is a part of a journal

• Title: International journal of legal medicine
• Shortened title: Int. j. leg. med.
• Publisher: Springer Nature
• ISSN: 1437-1596
• COBISS.SI-ID: 512020249

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: sekvenciranje naslednje generacije, Sangerjevo sekvenciranje, skeletni ostanki, druga svetovna vojna

Projects

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: J3-3080
• Name: Določitev predniškega porekla iz DNA za identifikacijske namene