An Application of ITO Analysis in Secondary Kinship Identification

Objective As the methods of the paternity and kinship testing have been developed, the second-degree and more distant relationships remain challenging in forensic science. Currently, the ITO method is the mainstream method to clarify the kinship between two individuals. Methods In this study, the ITO algorithm was used to calculate the uncle-nephew index based on 55 autosomal short tandem repeats (STRs) loci that were universally used for forensic identification. 19 STRs loci in Y chromosome were used for verification of the kinship. Results The cumulative uncle-nephew index between A and B was calculated to 0.993 by the analysis of the genotyping results of 21 STRs. When genotyping results of the other 34 STRs were added to the calculation algorithm, the cumulative uncle-nephew index between A and B was promoted to 227.928. Meanwhile, genotyping results of 17 Y-STRs loci showed that A and B shared the same Y-STRs haplotype that was in accord with the paternal inheritance law. Conclusion The biological uncle-nephew relationship between A and B are identified by applying the statistical principles and genetic technologies.


Introduction
In forensic genetics, short tandem repeats (STRs) on autosomes is the frequently-used genetic markers in the mainstream at present [1,2]. The stochastic ITO transition matrices provided by Li and Sacks in 1954 is a traditional methods to obtain the joint STR genotype distribution and genotypic correlations between any specified pair of noninbred relatives [3,4]. However, the sibling identification is complicated, and the conclusions risk uncertainties in some cases, because the potential intimate kinship between the two individuals involved in the kinship testing may limit the amount of genetic information that is usable for the identification. In this case study, an adult male (A) suspected that he might be abducted and trafficked into his current family at his early age. Because both the suspected father and the suspected mother died due to physical reasons and no material samples could be collected, the paternity identi-fication could not be conducted. So the man (A) requested an identification of the uncle-nephew kinship with his alive suspicious uncle (B). We applied the ITO algorithm using STRs in autosomes and Y chromosome in the identification of this secondary kinship. The validity of the ITO algorithm will provide more reliable evidences for the kinship identification.

Capillary Electrophoresis and Genotyping
Analysis. Capillary electrophoresis was performed using a 3130 XL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA), and genotype detection and analysis were performed using GeneMapper ID-X software (Thermo Fisher Scientific, Waltham, MA, USA).

Statistical Analysis.
The ITO method was applied to calculate the uncle-nephew index using genotyping data of 55 autosomal STRs loci, and the verification of the unclenephew kinship is carried using genotyping data of 17 Y-STRs loci.

Genotyping of Autosomal STRs
Loci. With the credible negative and positive quality controls, valid genotyping of total 55 STRs loci on autosomes in A and B was conducted. The genotyping results are shown in Table 1 Table 2. A and B shared the same genotype on all the 17 Y-STR loci.
3.3. Calculation for the Uncle-Nephew Index. The unclenephew index is calculated using the formula W = PI/ðPI + 1Þ. The cumulative uncle-nephew index between A and B was calculated to 0.993 by the analysis of the genotyping results of 21 STRs (from SiFaSTRTM 23 plex identification

Discussion
A complete description of the degree of relatedness of two individuals is a common and fundamental request in the forensic genetics [6]. In this case, there is an urgent demand for kinship identification without the genetic information of parents. However, there is no common standard for unclenephew kinship identification. So firstly, we used the genotyping results of 21 STRs from the conventional kit, SiFaSTRTM 23 plex identification system. The calculation method was revised according to the preliminary result, and the sufficient number of STRs loci was added. All STRs loci used in this study are all loci frequently used in forensic identification practice and have abundant population data which provide a reliable basis for the calculation reliability and the implementation feasibility. Meanwhile, when the uncle-nephew kinship was identified with relatively low index, the Y-STRs locus is a useful supplement in case that the tested individuals are all male [7]. The same Y-STRs haplotype found in the tested individuals enhance the reliability of the identification. This study calculated the uncle-nephew index using ITO method [3]. The ITO method is a classic way to identify the kinship between two individuals. Shao et al. pointed out that after the conclusion that shared alleles cannot be excluded from the analysis, ITO method can be further used to establish discriminant assumptions according to the specific case to obtain objective and reliable identification opinions [8]. In our study, it is demonstrated that when more autosomal STRs were used, the more uncle-nephew index between individuals A and B was obtained. It is demonstrated that the accuracy rate of the uncle-nephew kinship identification between two individuals increases with the number of the genetic markers. Therefore, more loci should be genotyped within the maximum testing capacity of the forensic laboratory's capacity for the complex kinship identifications, such as uncle-nephew relationships. However, the sufficient number of genetic marker loci and the rage of the probability value to verify the kinship in the complex cases should be investigated.
With the development of high-throughput sequencing and the establishment of genome database, kinship testing based on multiple genetic markers, such as SNPs and microhaplotypes, has great valuable practical applications [9,10]. In the future, we will apply more genetic markers, such as SNPs and indels, in solving complex kinship testing problems based on high-throughput sequencing [11,12].

Data Availability
The experimental data used to support the findings of this study are available from the corresponding author upon request.