A recommended sampling strategy for genetic identification of Second World War victims in Slovenia

Skeletonized human remains from Second World War mass graves in Slovenia are a major challenge in genetic identification, and bones with a high DNA yield must be selected for successful identification. The goal of this study was to construct skeletal sampling strategy recommendations through comparison of the most appropriate groups of skeletal elements. Altogether, 566 bones and teeth from the same mass grave were compared, half analyzed in this study and half in previous studies performed by our group. After anthropological examination, mechanical and chemical cleaning was performed, followed by bone and tooth powdering. Total demineralization of 0.5 g of bone and tooth was followed by extraction and purification of DNA with a Biorobot EZ1 device (Qiagen). The qPCR PowerQuant kit (Promega) was used to measure the amount of DNA, and statistical analysis was performed. Skeletal elements were selected according to known better preservation of DNA in the human body, and they were arranged in seven groups: petrous bone, long bones (femur and tibia), torso bones (first rib and 12th vertebra), metacarpals, metatarsals, short and sesamoid bones (talus, navicular, medial cuneiform, cuboid, calcaneus, and patella), and teeth. Sampling strategy recommendations were constructed based on DNA quantity and quality results. The petrous bone group, metacarpal group, torso bone group, and short and sesamoid bone group produced the highest DNA yields. Accordingly, in addition to standard sampling of long bones (femurs and tibias) and teeth, those additional bone types should be collected for Slovenian Second World War victim identification.