The set of 55 ancestry informative SNPs (AISNPs) originally developed by the Kidd Lab has been studied on a large number of populations and continues to be applied to new population samples. The existing reference database of population samples allows the relationships of new population samples to be inferred on a global level. Analyses show that these autosomal markers constitute one of the better panels of AISNPs. Continuing to build this reference database enhances its value. Because more than half of the 25 ethnic groups recently studied with these AISNPs are from Southwest Asia and the Mediterranean region, we present here various analyses focused on populations from these regions along with selected reference populations from nearby regions where genotype data are available. Many of these ethnic groups have not been previously studied for forensic markers. Data on populations from other world regions have also been added to the database but are not included in these focused analyses. The new population samples added to ALFRED and FROG-kb increase the total to 164 population samples that have been studied for all 55 AISNPs.
The interest in ancestry informative markers as an investigative tool to identify the biogeographical ancestry of potential suspects is increasing. For this purpose, many different panels have been designed to distinguish individuals from major geographical regions. The EUROFORGEN North Africa and Middle East ancestry panel is a second-tier panel designed for differentiating Middle Eastern/North African populations from other population groups. An Ion AmpliSeq™ custom panel (Thermo Fisher Scientific) was designed to sequence populations of interest using the Ion S5™ (Thermo Fisher Scientific). Of the 111 SNPs selected in the first version of the panel, 107 SNPs were successfully incorporated in the AmpliSeq™ multiplex PCR. More than 600 individuals from 13 different countries in the Middle East, North Africa, and Europe were typed. The performance of the sequencing assay was assessed by calculating relative locus balances, heterozygote balances, and noise levels. Lastly, likelihood ratios were calculated using the GenoGeographer software.
Ancestry informative markers have a great potential for ancestry inference in forensic genetic casework. However, applying ancestry inference in casework requires sufficient allele frequency data on as many populations as possible to allow the determination of biogeographical origin. Therefore, Turkish and Iranian individuals were SNP typed with Massively Parallel Sequencing with the Precision ID Ancestry Panel (Thermo Fisher Scientific) to assess whether it was possible to differentiate geographically proximate populations in the Middle East using this kit. Analyses showed that it were not possible to separate the two populations and neither was it possible to separate them from European and South-Central Asian populations. The two Middle Eastern populations were highly admixed with components from both Europe and South-Central Asia. Thus, it is important to identify genetic markers that are characteristic of the various Middle Eastern populations.
A custom GeneRead DNAseq SNP panel with 210 markers was evaluated using the Ion S5 and MiSeq sequencing platforms. Sensitivity, PCR cycle number, and the use of half volume of reagents for target enrichment and library preparation were tested. Furthermore, genotype concordance between results obtained with the different sequencing platforms and with known profiles generated using other sequencing assays was analysed. The GeneRead DNASeq SNP assay gave reproducible results with an input of 200 pg DNA on both platforms. A total of 204 loci were successfully sequenced. Three loci failed completely in the PCR amplification, and three additional loci displayed frequent locus drop-outs due to low read depth or high heterozygote imbalance. Overall, the read depth across the loci was more well-balanced with the MiSeq, while the heterozygote balance was less variable with the Ion S5. Noise levels were low on both platforms (median< 0.2 %). Two simple criteria for genotyping were applied: A minimum threshold of 45 reads and an acceptable heterozygote balance range of 0.3− 3.0. Complete concordance between platforms was observed except for three genotypes in one of the poorly performing loci, rs1470637. This locus had relatively low read depths on both platforms, skewed heterozygote balance, and frequent locus drop-outs. There was also full genotype concordance between the results from the GeneRead assay and known profiles generated with the QIAseq and Ion AmpliSeq assays. The few discordant results were either due to locus drop-outs in the poorly performing loci or allele drop-outs in the QIAseq assay. Profiles with a minimum of 179 SNPs were obtained from four challenging case work samples (blood swabs, bone, or blood from a corpse). Overall, the GeneRead DNASeq assay showed considerable potential and could provide a reliable method for SNP genotyping in cases involving identification of individuals, prediction of phenotypic traits, and ancestry inference.
Introduction of new methods requires meticulous evaluation before they can be applied to forensic genetic case work. Here, a custom QIAseq Targeted DNA panel with 164 ancestry informative markers was assessed using the MiSeq sequencing platform. Concordance, sensitivity, and the capability for analysis of mixtures were tested. The assay gave reproducible and nearly concordant results with an input of 10 and 2 ng DNA. Lower DNA input led to an increase in both locus and allele drop-outs, and a higher variation in heterozygote balance. Locus or allele drop-outs in the samples with less than 2 ng DNA input were not necessarily associated with the overall performance of a locus. Thus, the QIAseq assay will be difficult to implement in a forensic genetic setting where the sample material is often scarce and of poor quality. With equal or near equal mixture ratios, the mixture DNA profiles were easily identified by an increased number of imbalanced heterozygotes. For more skewed mixture ratios, the mixture DNA profiles were identified by an increased noise level. Lastly, individuals from Great Britain and the Middle East were investigated. The Middle Eastern individuals showed a greater affinity with South European populations compared to North European populations.
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