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

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.

Measure quantity of mitochondrial DNA in aged bones or calculate it from nuclear DNA quantitative PCR results?

Mitochondrial DNA (mtDNA) is of great value in forensics to procure information about a person when a next of kin, personal belongings, or other sources of nuclear DNA (nDNA) are unavailable, or nDNA is lacking in quality and quantity. The quality and reliability of the results depend greatly on ensuring optimal conditions for the given method, for instance, the optimal input of the copy number (CN) in next-generation sequencing (NGS) methods. The unavailability of commercial quantitative PCR (qPCR) methods to determine mtDNA CN creates the necessity to rely on recommendations to infer mtDNA CN from nDNA yield. Because nDNA yield varies between individuals, tissues, parts of the same tissue, and because mtDNA CN varies between tissues, such assumptions must be examined for a specific context, rather than be generalized. This study compares mtDNA CN calculated from nDNA yield and qPCR measured mtDNA CN. Seventy-five femurs from the Second World War victims were used as samples; they were cut below the greater trochanter, surface contaminants were removed by mechanical and chemical cleaning, samples were fully demineralized, and DNA was isolated. PowerQuant® Kit (Promega) was used to analyze DNA yield. An in-house method was used to determine mtDNA CN. Comparison of mtDNA CN from nDNA derived calculations and measured mtDNA CN highlighted vast differences. The results emphasize the need to perform qPCR to assess mtDNA CN before NGS analyses of aged bones' mitogenomes rather than estimating mtDNA CN from nDNA yield to ensure the quality and reliability of the results of NGS analysis.

High DNA yield from metatarsal and metacarpal bones from Slovenian Second World War skeletal remains.

DNA yield varies by anatomical region, and the selection of bone types that yield maximum recovery of DNA is important to maximize the success of human identification of skeletal remains. The goal of our study was to explore inter- and intra-individual variation in DNA content by measuring nuclear DNA quantity and quality and autosomal STR typing success to determine the most promising skeletal elements for bone sampling. To exclude the influence of taphonomic issues as much as possible, three complete male skeletons from a single Second World War mass grave were examined and all representative skeletal element types of the human body were analyzed. Forty-eight different types of bones from the head, torso, arm, leg, hand, and foot were sampled from each skeleton, 144 bones altogether. The samples were cleaned, and half a gram of bone powder was decalcified using a full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen). DNA content and rates of DNA degradation were determined with the PowerQuant (Promega), and the Investigator ESSplex SE QS (Qiagen) was used for STR typing. The highest-yielding bones mostly produced the most complete STR profiles. Among the skeletal elements containing on average the most DNA and producing the most complete profiles in all three skeletons examined were metacarpals, metatarsals, and the petrous portion of the temporal bone. Metatarsals and metacarpals can easily be sampled without using a saw, thus reducing potential DNA contamination. Skeletons from the Second World War can be used as a model for poorly preserved skeletal remains, and the results of the investigation can be applied for genetic identification of highly degraded skeletal remains in routine forensic casework. Although the research was limited to only three skeletons found in a unique mass grave, the data obtained could contribute to sampling strategies for identifying old skeletal remains. More Second World War skeletons will be analyzed in the future to investigate inter-bone variation in the preservation of DNA.

Identifying victims of the largest Second World War family massacre in Slovenia.

The killings during the Second World War, with nearly one hundred thousand victims, is one of the greatest losses of life in Slovenia's modern history. This article presents the genetic identification of the victims of the largest family massacre that occurred in Slovenia, in which 10 members of the same family were killed. Seven of them were buried in a hidden mass grave and only two children survived. In 2015 and 2016, two graves were found and three incomplete female skeletons and at least three incomplete male skeletons were exhumed. A total of 12 bones and teeth were analysed and compared to two living relatives. Extracted DNA was quantified using the PowerQuant kit, and various autosomal and Y-STR kits were used for STR typing. Up to 2.7 ng DNA/g of powder was acquired from the samples analysed. We managed to obtain nuclear DNA for successful STR typing from seven bones and one molar. From the female grave, autosomal profiles were obtained only from one skeleton, and from the male grave from five out of six femurs. The relationships between the males were additionally confirmed by analyses of Y-STRs. STR profiles made possible the identification of four family members; one of the aunts from the female grave, and two uncles and the father of the surviving children, who were used as family references, from the male grave. The product rule was used to calculate a combined likelihood ratio for autosomal and Y-STRs, and statistical analyses showed high confidence of correct identification with posterior probability (PP) greater than 99.9 % for three out of four victims identified. For identifying the aunt, the PP obtained after ESI-17 and NGM STR typing was too low. To increase the PP, the next-generation sequencing Precision ID GlobalFiler NGS STR Panel was used and, after the analysis of additional STR loci, the statistical analysis showed a PP greater than 99.9 %, indicating that a sufficient number of genetic markers had been investigated in identifying the skeletal remains of the aunt. An elimination database containing the genetic profiles of all individuals that had been in contact with the bones was created to ensure traceability in case of contamination, and no matches were found. After more than 70 years, the skeletal remains were returned to the surviving children, who buried their relatives in a family grave.