Evaluation of commercially available DNA testing kits to resolve complex kinship disputes in a South African setting

Background: Human identification techniques using biological material have been under development for over a century. By the early 2000s, routine DNA profiling, using short tandem repeats (STRs), was in place as a standard of practice in both forensic science and in parentage testing laboratories. Use of the commercial kits, containing less than 20 STR loci, led to inconclusive results in certain cases, particularly when kinship testing had to be performed in the absence of one or both parents. It is proposed that better resolution can be achieved when more STR loci are used to calculate Likelihood Ratios (LRs) for establishing biological relationships. Recently, newer kits containing more than 20 STR loci, have become available. The present study aimed to look at the utility of two new commercial kits with expanded numbers of STR loci and to assess the validity of outcomes when fewer loci were utilized for assessment of complex kinship queries, in a large parentage testing laboratory within the National Health Service in South Africa. Further, the study aimed to evaluate different software packages available for performing the statistical calculations associated with this genetic testing approach. Subjects and methods: A total of 729 samples, collected from unrelated individuals from seven ethnic groups, namely: South African Bantu-speakers (n=366), South African Whites (n=84), South African mixed ancestry (Coloureds) (n=40), South African Indians (n=26), Zimbabwean Bantu-speakers (n=113), Nigerian Bantu-speakers (n=73) and Namibian Bantu-speakers (n=27), were used in the present study. STR-based polymerase chain reaction (PCR) amplification was performed using either the PowerPlex® Fusion system (Promega Corporation) or the VeriFiler™ Express PCR amplification kit (Life Technologies), both of which contain more than 20 STR loci, compared to the DNA Profiling Laboratory (DPL)’s currently used commercial kits which contain 16 or 18 STR loci. Allele frequencies were calculated for each ethnic group where n>40, using Microsoft Excel® 2016 (counting method). Hardy-Weinberg Equilibrium (HWE), linkage disequilibrium (LD) and population genetic variation tests were performed using GenePop. Kinship queries, especially those with inconclusive outcomes when 15 autosomal STR loci were used in the initial, routine testing, were retested using the new expanded kits. Outcomes obtained from using four different analysis software packages, namely, the In-house Microsoft Excel macro-enabled software, Converge™ Software, GeneMarker® HID and Familias were compared to assess if there was a statistical difference in the outcomes obtained when calculating LRs using these different analysis tools. Results: The genotype results showed that the probability of identity (PI) increased as the number of STR loci contained in a commercial kit increased. Thus, a significantly higher discrimination power was achieved when the PowerPlex® Fusion system and the VeriFiler™ Express PCR kit were used, as compared to the routinely used kits which contain 16 or 18 STR loci. More than 50% of cases with initially inconclusive outcomes, when retested with expanded kits, could now be resolved based on additional STR loci. Populations investigated in the present study were found to be in HWE when assessed for heterozygosity excess. Departures from HWE were detected in the SA and Nigerian Bantu-speakers, when assessed for heterozygosity deficiency. Linkage disequilibrium was observed (erroneously) between loci located on different chromosomes. Genetic diversity tests (Fst) showed that the SA and Zimbabwean Bantu-speakers can be clustered together, and the same allele frequencies can be used for calculating LRs involving these two groups. The analysis software packages evaluated all gave p-values > 0.05 when compared to each other, indicating that there was no significant difference in the Likelihood Ratios obtained when different analysis software packages were used Conclusion: The inclusion of additional STR loci in the newer commercial kits for human identity testing was shown to be beneficial in kinship analysis. It is recommended that all kinship queries should be tested using the newer kits as a first line testing approach. Additional testing, using lineage markers such as X and Y chromosomes and mitochondrial DNA, when applicable, is recommended as part of first line testing, to reduce long turn-around times and ensure conclusive outcomes. The In-house data analysis software package currently used in DPL was validated for routine use
A dissertation submitted in fulfilment of the requirements for the degree of Master of Science in Medicine to the Faculty of Health Sciences, School of Pathology, University of the Witwatersrand, Johannesburg, 2020