Comparison of the optimal and suboptimal quantity of mitotype libraries using next-generation sequencing

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

Optimizing analysis parameters and sample input is crucial in forensic genetics methods to generate reliable results, and even more so when working with muti-copy mitochondrial DNA (mtDNA) and low-quality samples. This study compared mitotypes based on next-generation sequencing (NGS) results derived from the same samples at two different sequencing library concentrations—30 pM and 0.3 pM. Thirty femur samples from the Second World War were used as a model for poorly preserved DNA. Quantitative PCR (qPCR) method targeting 113 bp long fragment was employed to assess the quantity of mitogenomes. HID Ion Chef™ Instrument with Precision ID mtDNA Control Region Panel was used for library preparation and templating. Sequencing was performed with Ion GeneStudio™ S5 System. Reference haplotypes were determined from sequencing samples at 30 pM library input. Haplotypes were compared between optimal (30 pM) and suboptimal (0.3 pM) library inputs. Often the difference in haplotypes was length heteroplasmy, which in line with other studies shows that this type of variant is not reliable for interpretation in forensics. Excluding length variants at positions 573, 309, and 16,193, 56.7% of the samples matched, and in two samples, no sequence was obtained at suboptimal library input. The rest of the samples differed between optimal and suboptimal library input. To conclude, genotyping and analyzing low-quantity libraries derived from low-quality aged skeletonized human remains therefore must be done with caution in forensic genetics casework.

• Language: English
• Keywords: haplotype, library input, mitogenome, next-generation sequencing, Second World War
• Work type: Article
• Typology: 1.03 - Other scientific articles
• Organization: MF - Faculty of Medicine
• Publication status: Published
• Publication version: Version of Record
• Year: 2024
• Number of pages: Str. 395–400
• Numbering: Vol. 138, iss. 2
• PID: 20.500.12556/RUL-154409
• UDC: 616:340.6
• ISSN on article: 1437-1596
• DOI: 10.1007/s00414-023-03099-7
• COBISS.SI-ID: 167475971
• Publication date in RUL: 13.02.2024

OBAL, Marcel, ZUPANC, Tomaž and ZUPANIČ-PAJNIČ, Irena, 2024, Comparison of the optimal and suboptimal quantity of mitotype libraries using next-generation sequencing. International journal of legal medicine [online]. 2024. Vol. 138, no. 2. [Accessed 13 August 2025]. DOI 10.1007/s00414-023-03099-7. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=154409

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: haplotip, vnos v knjižnico, mitogenom, sekvenciranje naslednje generacije, druga svetovna vojna

Projects

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