Determining family relationships is a fundamental application of forensic genetics and is commonly performed using STRs, which are well suited for close kinship analysis. However, their relatively high mutation rates and limited abundance in the genome reduce their reliability for more distant relationships. In contrast, SNPs exhibit lower mutation rates but typically require larger numbers to provide information equivalent to a single STR marker, increasing sequencing complexity and analytical costs. Microhaplotypes (MHs), short genomic regions comprising several closely linked SNPs, combine high polymorphism with low mutation rates and therefore represent a promising marker type for extended kinship analysis. However, most available MH panels still comprise relatively few markers, and sufficiently large panels for extended kinship inference remain scarce.

To address this gap, we developed the MIcrohaplotype panel for Extended Kinship Analyses (MIEKA), comprising 286 microhaplotypes compiled from eight existing MH panels. The MIEKA panel was designed to enhance discrimination between full siblings (FS) and half siblings (HS) while also including the potential to resolve third-degree relationships. Performance of the MIEKA panel, alone and in combination with 32 STRs, was evaluated using simulated kinship pairs (first to fifth-degree) generated with FamLink2, and compared with STR- and SNP-based panels. Likelihood ratios for FS versus HS were calculated using Merlin based on 100,000 simulated pairs per relationship type. Robustness was assessed through a sensitivity assay and simulations of locus and allelic dropout. Applicability of the panel to low DNA quality/quantity was examined using historical samples from two families with known family trees. In addition, three recent high-quality forensic casework samples were included.

The combined MIEKA and STR panel provides improved resolution for relationships up to the third degree, whereas discrimination beyond the fourth degree is no longer reliable. More than 99% of FS and HS pairs were correctly classified, with only one misclassification among the 200,000 simulated pairs. Classification remained robust under locus dropout, while allelic dropout above 30% led to a gradual increase in misclassifications. Genotype profiles were obtained for 12 out of 14 historical samples using MIEKA, and the MIEKA+STR panel successfully resolved all tested relationship constellations up to the third degree. In all three forensic cases, the MIEKA+STR panel provided clear discrimination between the competing hypotheses of FS and HS.

Overall, MIEKA bridges the gap between STRs and large SNP panels, providing a versatile tool for extended kinship analysis and enabling robust FS and HS discrimination even in challenging samples.

Authors

• Theresa Roth (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)
• Joelle Schneider (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)
• Peter Resutik (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)
• Mario Gysi (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)
• Daniel Kling (Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Sweden)
• Cordula Haas (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)
• Natasha Arora (Zurich Institute of Forensic Medicine, University of Zurich, Switzerland)