Design, installation, and performance evaluation of a custom dye matrix standard for automated capillary electrophoresis.

CE equipment detects and deconvolutes mixtures containing up to six fluorescently labeled DNA fragments. This deconvolution is done by the collection software that requires a spectral calibration file. The calibration file is used to adjust for the overlap that occurs between the emission spectra of fluorescence dyes. All commercial genotyping and sequencing kits require the installation of a corresponding matrix standard to generate a calibration file. Due to the differences in emission spectrum overlap between fluorescent dyes, the application of existing commercial matrix standards to the electrophoretic separation of DNA labeled with other fluorescent dyes can yield undesirable results. Currently, the number of fluorescent dyes available for oligonucleotide labeling surpasses the availability of commercial matrix standards. Therefore, in this study we developed and evaluated a customized matrix standard using ATTO 633, ATTO 565, ATTO 550, ATTO Rho6G, and 6-FAM dyes for which no commercial matrix standard is available. We highlighted the potential genotyping errors of using an incorrect matrix standard by evaluating the relative performance of our custom dye set using six matrix standards. The specific performance of two genotyping kits (UniQTyper™ Y-10 version 1.0 and PowerPlex® Y23 System) was also evaluated using their specific matrix standards. The procedure we followed for the construction of our custom dye matrix standard can be extended to other fluorescent dyes.

Evaluation of the InnoTyper® 21 genotyping kit in multi-ethnic populations.

We report the findings of the evaluation of the InnoTyper® 21 genotyping kit for the use of human identification (HID) and paternity testing in South Africa. This novel forensic kit evaluates 20 retrotransposable elements (AC4027, MLS26, ALU79712, NBC216, NBC106, RG148, NBC13, AC2265, MLS09, AC1141, TARBP, AC2305, HS4.69, NBC51, ACA1766, NBC120, NBC10, NBC102, SB19.12 and NBC148) and the Amelogenin locus for sex determination. The evaluation of the genotyping performance showed no significant spectral pull-up for peak heights between 100 and 30,000 RFUs. All loci presented biallelic patterns except the triallelic RG148 locus resulting from a variant insertion allele, named RG148I-1, observed exclusively in the Bantu. The InnoTyper® 21 kit was found to be highly discriminatory between the 507 unrelated individuals of the Afrikaaner, Asian Indian, Coloured, amaXhosa and amaZulu groups. The HID parameters: the CPD ranged between 0.99999987 and 0.9999999845, and the CMP between 1.0335×10-7 and 1.5506×10-8. The paternity parameters: the CPI ranged between 0.0202 and 0.3177, and the CPE between 0.9161 and 0.9749. There were no significant signs of deviations from HWE or linkage disequilibrium (LD) after applying a Bonferroni correction. This kit also showed minor levels of population structure which could differentiate between the African and non-African population groups. Finally, in challenging casework with severely degraded biological material, the InnoTyper® 21 genotyping kit was compatible with GlobalFiler® and Investigator DIPplex® to increase the HID parameters.

GlobalFiler(®) Express DNA amplification kit in South Africa: Extracting the past from the present.

In this study, the GlobalFiler(®) Express amplification kit was evaluated for forensic use in 541 South African individuals belonging to the Afrikaaner, amaXhosa,(1) amaZulu,(1) Asian Indian and Coloured population groups. Allelic frequencies, genetic diversity parameters and forensic informative metrics were calculated for each population. A total of 301 alleles were observed ranging between 5 and 44.2 repeat units, 43 were rarely observed partial repeats and seven were novel. The combined match probability (CMP) ranged from 2.21×10(-26) (Coloured) to 5.21×10(-25) (AmaZulu), and the combined power of exclusion (CPE) 0.9999999978 (Afrikaaner) to 0.99999999979 (AmaZulu) respectively. No significant departures from Hardy-Weinberg equilibrium (HWE) were observed after Bonferroni correction. Strong evidence of genetic structure was detected using the coancestry coefficient θ, Analysis of Molecular Variance (AMOVA) and an unsupervised Bayesian clustering method (STRUCTURE). The efficiency of assignment of individuals to population groups was evaluated by applying likelihood ratios with WHICHRUN, and the individual ancestral membership probabilities inferred by STRUCTURE. Likelihood ratios performed the best in the assignment of individuals to population groups. Signs of positive selection were detected for TH01 and D13S317 and purifying/balancing selection for locus SE33. These three loci also displayed the largest informativeness for assignment (In) values. The results of this study supports the use of the GlobalFiler(®) STR profiling kit for forensic applications in South Africa with the additional capability to predict ethnicity or continental origin of a random sample.

Where is the game? Wild meat products authentication in South Africa: a case study.

BACKGROUND: Wild animals' meat is extensively consumed in South Africa, being obtained either from ranching, farming or hunting. To test the authenticity of the commercial labels of meat products in the local market, we obtained DNA sequence information from 146 samples (14 beef and 132 game labels) for barcoding cytochrome c oxidase subunit I and partial cytochrome b and mitochondrial fragments. The reliability of species assignments were evaluated using BLAST searches in GenBank, maximum likelihood phylogenetic analysis and the character-based method implemented in BLOG. The Kimura-2-parameter intra- and interspecific variation was evaluated for all matched species. RESULTS: The combined application of similarity, phylogenetic and character-based methods proved successful in species identification. Game meat samples showed 76.5% substitution, no beef samples were substituted. The substitutions showed a variety of domestic species (cattle, horse, pig, lamb), common game species in the market (kudu, gemsbok, ostrich, impala, springbok), uncommon species in the market (giraffe, waterbuck, bushbuck, duiker, mountain zebra) and extra-continental species (kangaroo). The mountain zebra Equus zebra is an International Union for Conservation of Nature (IUCN) red listed species. We also detected Damaliscus pygargus, which is composed of two subspecies with one listed by IUCN as 'near threatened'; however, these mitochondrial fragments were insufficient to distinguish between the subspecies. The genetic distance between African ungulate species often overlaps with within-species distance in cases of recent speciation events, and strong phylogeographic structure determines within-species distances that are similar to the commonly accepted distances between species. CONCLUSIONS: The reliability of commercial labeling of game meat in South Africa is very poor. The extensive substitution of wild game has important implications for conservation and commerce, and for the consumers making decisions on the basis of health, religious beliefs or personal choices.Distance would be a poor indicator for identification of African ungulates species. The efficiency of the character-based method is reliant upon availability of large reference data. The current higher availability of cytochrome b data would make this the marker of choice for African ungulates. The encountered problems of incomplete or erroneous information in databases are discussed.