Improving kinship probability in analysis of ancient skeletons using identity SNPs and MPS technology

In forensic kinship analysis and human identification cases, analysis of STRs is the gold standard. When badly preserved ancient DNA is used for kinship analysis, short identity SNPs are more promising for successful amplification. In this work, kinship analysis was performed on two skeletons from the Early Middle Ages. The surface contaminants of petrous bones were removed by chemical cleaning and UV irradiation; DNA was isolated through full demineralization and purified in an EZ1 Advanced XL machine. The PowerQuant kit was used to analyze DNA yield and degradation, and on average, 17 ng DNA/g of petrous bone was obtained. Both skeletons were typed in duplicate for STR markers using the Investigator EssplexPlus SE QS kit, and comparison of partial consensus genotypes showed shared allelic variants at most loci amplified, indicating close kinship. After statistical calculation, the full-sibling kinship probability was too low for kinship confirmation, and additional analyses were performed with PCR-MPS using the Precision ID Identity Panel. The HID Ion Chef Instrument was used to prepare the libraries and for templating and the Ion GeneStudio S5 System for sequencing. Analysis of identity SNPs produced full genetic profiles from both skeletons. For combined likelihood ratio (LR) calculation, the product rule was used, combining LR for STRs and LR for SNPs, and a combined LR of 3.3 × 10⁷ (corresponding to a full-sibling probability of 99.999997%) was calculated. Through the SNP PCR-MPS that followed the STR analysis, full-sibling kinship between the ancient skeletons excavated from an early medieval grave was confirmed.