The development of the novel 22 X- indel multiplex system for forensic genetics.

InDel (Insertions/deletion) markers have been used as an alternative, or as a complementary marker system, to STR markers in human identification due to their advantages such as low mutation rates, no stutter, and potential small amplicon sizes. In forensic sciences, sex chromosomes are widely used in forensic genetics for specific cases. For example, the relationship between father and daughter can be determined by using X-InDels. In this study, we developed a novel 22 X-InDel multiplex system that was identified by two separate assays with fluorescence amplification and capillary electrophoresis detection technology. We chose 22 X-InDel markers based on the following criteria: mean heterozygosity over 30% in Europeans; minimum of 250 Kb differences between each InDel loci; and an amplicon length that was less than 300 bp. We performed an optimization and validation study of 22 X-InDel systems under the following parameters: analytical threshold, sensitivity, precision and accuracy, stochastic threshold, repeatability, and reproducibility. We evaluated the allele frequency of this multiplex system in the Turkish population, and then the population comparisons were carried out on data from 1000 Genome populations (Europe, Africa, America, South Asia, and East Asia). The sensitivity test showed a complete genotyping profile with DNA concentrations as low as 0.5 ng. The heterozygosity ratio of 22 X-InDel loci was determined as 0.4690 and the discrimination power was defined as 0.99. The results show that the new 22 X-InDel multiplex system provides high polymorphism information, and it is a reproducible, accurate, sensitive, and robust system that could be used as an additional tool for kinship testing.