Molecular genetic identification of skeletal remains from the Second World War Konfin I mass grave in Slovenia

This paper describes molecular genetic identification of one third of the skeletal remains of 88 victims of postwar (June 1945) killings found in the Konfin I mass grave in Slovenia. Living relatives were traced for 36 victims. We analyzed 84 right femurs and compared their genetic profiles to the genetic material of living relatives. We cleaned the bones, removed surface contamination, and ground the bones into powder. Prior to DNA isolation using Biorobot EZ1 (Qiagen), the powder was decalcified. The nuclear DNA of the samples was quantified using the real-time polymerase chain reaction method. We extracted 0.8 to 100 ng DNA/g of bone powder from 82 bones. Autosomal genetic profiles and Y-chromosome haplotypes were obtained from 98% of the bones, and mitochondrial DNA (mtDNA) haplotypes from 95% of the bones for the HVI region and from 98% of the bones for the HVII region. Genetic profiles of the nuclear and mtDNA were determined for reference persons. For traceability in the event of contamination, we created an elimination database including genetic profiles of the nuclear and mtDNA of all persons that had been in contact with the skeletal remains. When comparing genetic profiles, we matched 28 of the 84 bones analyzed with living relatives (brothers, sisters, sons, daughters, nephews, or cousins). The statistical analyses showed a high confidence of correct identification for all 28 victims in the Konfin I mass grave (posterior probability ranged from 99.9% to more than 99.999999%)

MPS reveals isometric PCR artefacts in degraded samples

It is well known that DNA damage promotes PCR artefacts. In addition, as MPS provides the most accurate typing of PCR products, this technology should be able to highlight more PCR artefacts than the conventional CE approach. To test this hypothesis, a DNA sample was degraded by heating at 70 \ub0C for 8, 16 and 24 h. The three resulting samples #1, #2 and #3 and the untreated control sample were then analysed by the Precision ID Globalfiler NGS STR Panel v2, in duplicate tests. The data analysis was performed by the Converge v2.1 software using the default setting parameters. Artefacts were identified only in the degraded samples #2 and #3. Few well known artefacts (allelic imbalance, stutter products and allelic drop out) and several drop in phenomena were flagged by the software. The majority (18 out 22) of these drop ins were sequenced as \u201cisometric artefacts\u201d, e.g. PCR artefacts which cannot be identified by the employment of CE. The molecular features of these PCR artefacts, as well as their risk to be confused with a DNA mixture, are described

Technical note: On the long term storage of forensic DNA in water

A rectrospective study was conducted on the effect of the long term storage of 122 DNA samples resuspended in water, one of the elution media still suggested by well established protocols. These DNA samples come from four different kinds of forensically relevant samples (saliva swabs, FTA card bloodstains, nails and II\ub0 World War bones) extracted in 2008-2018 and stored at \u2013 20 \ub0C (n=113 of groups #1-#5) and at + 4 \ub0C (n=9 of the group #6), respectively. At the time of the present study (2019), quantitative PCR (qPCR) was employed as tool for assessing the degradation of the samples. The employment of the Human Quantifiler Kit showed that the median loss of DNA ranged from 17.8% to 66.6 % in groups #1-#5 while it was 85.0 % in group #6. However, it is likely that these values represent an underestimation due to the shortness of the qPCR probe (62 bp). Noteworthy, the DNA loss was statistically significant in each of the six groups (p values 64 0.0167). Thus, in agreement with the data on spontaneous DNA decay, no forensic DNA sample should be stored in water for long term periods. In conclusion, the results of this technical note warn against the use of water for this purpose