Systematically improving the efficiency of complex kinship identification through the addition of genetic markers and reference individuals.

There is a growing demand for complex kinship identification due to the increasing complexity of social structures. However, the system efficiency of complex kinship identification systems, particularly for identifying second-degree and higher kinship relationships, needs improvement. This study aimed to further explore the selection of introduced relatives and genetic markers for second- and third-degree kinship identification by comparing the statistical parameters, such as likelihood ratio (LR), effectiveness, and error rate, of four different detection systems (19 STRs, 39 STRs, 55 STRs, and 55 STRs + 94 SNPs) in 109 common kinship identification scenarios. The study found that increasing the number of genetic markers or introducing more reference individuals generally enhanced the system efficiency for identifying complex genetic relationships by introducing various reference individuals into three-generation family lines to identify second- and third-degree kinships. The results were validated using true pedigree samples. Ranking the system efficiencies of kinship identification systems with different combinations of reference individuals and genetic markers provided insight into reference selection priorities and the optimal number of loci required. For second-degree relatives, adding one relative to the 39 STR marker set resulted in an effectiveness of over 90% with an error rate of less than 0.0005, while adding two known relatives achieved an effectiveness of over 95% with an error rate of less than 0.0001. Certain models could completely discriminate between related and unrelated individuals when using 55 STRs. For third-degree relatives, incorporating two known relatives in the 55 STR marker set resulted in an effectiveness of over 90% with an error rate of less than 0.0001 in most scenarios. In addition, when using both 55 STRs and 94 SNPs, a total of five models used for first-cousin relationships, that incorporated two reference individuals within second-degree relatives of both subjects, could completely distinguish between related and unrelated individuals. This study not only offers theoretical insights for complex kinship identification but also provides a valuable reference that can significantly improve the practice of identifying second- and third-degree kinships.