Exploring iris colour prediction and ancestry inference in admixed populations of South America

New DNA-based predictive tests for physical characteristics and inference of ancestry are highly informative tools that are being increasingly used in forensic genetic analysis. Two eye colour prediction models: a Bayesian classifier – Snipper and a multinomial logistic regression (MLR) system for the Irisplex assay, have been described for the analysis of unadmixed European populations. Since multiple SNPs in combination contribute in varying degrees to eye colour predictability in Europeans, it is likely that these predictive tests will perform in different ways amongst admixed populations that have European co-ancestry, compared to unadmixed Europeans. In this study we examined 99 individuals from two admixed South American populations comparing eye colour versus ancestry in order to reveal a direct correlation of light eye colour phenotypes with European co-ancestry in admixed individuals. Additionally, eye colour prediction following six prediction models, using varying numbers of SNPs and based on Snipper and MLR, were applied to the study populations. Furthermore, patterns of eye colour prediction have been inferred for a set of publicly available admixed and globally distributed populations from the HGDP-CEPH panel and 1000 Genomes databases with a special emphasis on admixed American populations similar to those of the study samples.     

Analysis of ancestry informative markers in three main ethnic groups from Ecuador supports a trihybrid origin of Ecuadorians

Ancestry inference is traditionally done using autosomal SNPs that present great allele frequency differences among populations from different geographic regions. These ancestry informative markers (AIMs) are useful for determining the most likely biogeographic ancestry or population of origin of an individual. Due to the growing interest in AIMs and their applicability in different fields, commercial companies have started to develop AIM multiplexes targeted for Massive Parallel Sequencing platforms.This project focused on the study of three main ethnic groups from Ecuador (Kichwa, Mestizo, and Afro-Ecuadorian) using the Precision ID Ancestry panel (Thermo Fisher Scientific). In total, 162 Ecuadorian individuals were investigated. The Afro-Ecuadorian and Mestizo showed higher average genetic diversities compared to the Kichwa. These results are consistent with the highly admixed nature of the first two groups. The Kichwa showed the highest proportion of Native Amerindian (NAM) ancestry relative to the other two groups. The Mestizo had an admixed ancestry of NAM and European with a larger European component, whereas the Afro-Ecuadorian were highly admixed presenting proportions of African, Native Amerindian, and European ancestries. The comparison of our results with previous studies based on uniparental markers (i.e. Y chromosome and mtDNA) highlighted the sex-biased admixture process in the Ecuadorian Mestizo.Overall, the data generated in this work represent one important step to assess the application of ancestry inference in admixed populations in a forensic context.     

Development and validation of the EUROFORGEN NAME (North African and Middle Eastern) ancestry panel

Inference of biogeographic origin is an important factor in clinical, population and forensic genetics. The information provided by AIMs (Ancestry Informative Markers) can allow the differentiation of major continental population groups, and several AIM panels have been developed for this purpose. However, from these major population groups, Eurasia covers a wide area between two continents that is difficult to differentiate genetically. These populations display a gradual genetic cline from West Europe to South Asia in terms of allele frequency distribution. Although differences have been reported between Europe and South Asia, Middle East populations continue to be a target of further investigations due to the lack of genetic variability, therefore hampering their genetic differentiation from neighboring populations. In the present study, a custom-built ancestry panel was developed to analyze North African and Middle Eastern populations, designated the ‘NAME’ panel. The NAME panel contains 111 SNPs that have patterns of allele frequency differentiation that can distinguish individuals originating in North Africa and the Middle East when combined with a previous set of 126 Global AIM-SNPs.     

Building a forensic ancestry panel from the ground up: The EUROFORGEN Global AIM-SNP set

Emerging next-generation sequencing technologies will enable DNA analyses to add pigmentation predictive and ancestry informative (AIM) SNPs to the range of markers detectable from a single PCR test. This prompted us to re-appraise current forensic and genomics AIM-SNPs and from the best sets, to identify the most divergent markers for a five population group differentiation of Africans, Europeans, East Asians, Native Americans and Oceanians by using our own online genome variation browsers. We prioritized careful balancing of population differentiation across the five group comparisons in order to minimize bias when estimating co-ancestry proportions in individuals with admixed ancestries. The differentiation of European from Middle East or South Asian ancestries was not chosen as a characteristic in order to concentrate on introducing Oceanian differentiation for the first time in a forensic AIM set. We describe a complete set of 128 AIM-SNPs that have near identical population-specific divergence across five continentally defined population groups. The full set can be systematically reduced in size, while preserving the most informative markers and the balance of population-specific divergence in at least four groups. We describe subsets of 88, 55, 28, 20 and 12 AIMs, enabling both new and existing SNP genotyping technologies to exploit the best markers identified for forensic ancestry analysis.     

A single nucleotide polymorphism panel for individual identification and ancestry assignment in Caucasians and four East and Southeast Asian populations using a machine learning classifier

Single nucleotide polymorphism (SNP) profiling is an effective means of individual identification and ancestry inferences in forensic genetics. This study established a SNP panel for the simultaneous individual identification and ancestry assignment of Caucasian and four East and Southeast Asian populations. We analyzed 220 SNPs (125 autosomal, 17 X-chromosomal, 30 Y-chromosomal, and 48 mitochondrial SNPs) of the DNA samples from 563 unrelated individuals of five populations (89 Caucasian, 234 Taiwanese Han, 90 Filipino, 79 Indonesian and 71 Vietnamese) and 18 degraded DNA samples. Informativeness for assignment (In) was used to select ancestry informative SNPs (AISNPs). A machine learning classifier, support vector machine (SVM), was used for ancestry assignment. Of the 220 SNPs, 62 were individual identification SNPs (IISNPs) (51 autosomal and 11 X-chromosomal SNPs) and 191 were AISNPs (100 autosomal, 13 X-chromosomal, 30 Y-chromosomal, and 48 mitochondrial SNPs). The 51 autosomal IISNPs offered cumulative random match probabilities (cRMPs) ranging from 1.56 × 10−21 to 3.16 × 10−22 among these five populations. Using AISNPs with the SVM, the overall accuracy rate of ancestry inference achieved in the testing dataset between Caucasian, Taiwanese Han, and Filipino populations was 88.9%, whereas it was 70.0% between Caucasians and each of the four East and Southeast Asian populations. For the 18 degraded DNA samples with incomplete profiling, the accuracy rate of ancestry assignment was 94.4%. We have developed a 220-SNP panel for simultaneous individual identification and ethnic origin differentiation between Caucasian and the four East and Southeast Asian populations. This SNP panel may assist with DNA analysis of forensic casework.     

Development and inter-laboratory evaluation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA

Responding to the growing scientific and practical interest in forensic DNA phenotyping, the VISible Attributes through GEnomics (VISAGE) Consortium was founded in 2017 with the main goal of developing and validating new and reliable molecular and statistical tools to predict appearance, ancestry and age from DNA. Here, we describe the development and inter-laboratory evaluation and validation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA. The VISAGE Enhanced Tool for Appearance and Ancestry is the first forensic-driven genetic laboratory tool that comprises well-established markers for eye, hair and skin color with more recently discovered DNA markers for eyebrow color, freckling, hair shape and male pattern baldness and bio-geographic ancestry informative DNA markers. The bio-geographic ancestry markers include autosomal SNPs (bi- and tri-allelic SNPs), X-SNPs, Y-SNPs and autosomal Microhaplotypes. In total, primers targeting 524 SNPs (representing a 97.6% assay conversion rate) were successfully designed using AmpliSeq into a single primer pool (i.e., one multiplex assay) and sequenced with the Ion S5. In a collaborative framework, five VISAGE laboratories tested the VISAGE Enhanced Tool for Appearance and Ancestry on reproducibility, sensitivity, genotyping concordance, mixtures, species specificity and performance in relevant forensic conditions, including inhibitor-spiked, mock casework and artificially degraded samples. Based on our results, the VISAGE Enhanced Tool for Appearance and Ancestry is a robust, reproducible, and – for the large SNP number - fairly sensitive MPS assay with high concordance rates. With the VISAGE Enhanced Tool for Appearance and Ancestry introduced here, the VISAGE Consortium delivers the first single DNA-test for combined appearance prediction based on seven traits together with bio-geographic ancestry inference based on major continental regions for separated bi-parental and paternal ancestry, which represents the most comprehensive validated laboratory tool currently available for Forensic DNA Phenotyping.     

The Global AIMs Nano set: A 31-plex SNaPshot assay of ancestry-informative SNPs

A 31-plex SNaPshot assay, named ‘Global AIMs Nano’, has been developed by reassembling the most differentiated markers of the EUROFORGEN Global AIM-SNP set. The SNPs include three tri-allelic loci and were selected with the goal of maintaining a balanced differentiation of: Africans, Europeans, East Asians, Oceanians and Native Americans. The Global AIMs Nano SNP set provides higher divergence between each of the five continental population groups than previous small-scale AIM sets developed for forensic ancestry analysis with SNaPshot. Both of these characteristics minimise potential bias when estimating co-ancestry proportions in individuals with admixed ancestry; more likely to be observed when using markers disproportionately informative for only certain population group comparisons. The optimised multiplex is designed to be easily implemented using standard capillary electrophoresis regimes and has been used to successfully genotype challenging forensic samples from highly degraded material with low level DNA. The ancestry predictive performance of the Global AIMs Nano set has been evaluated by the analysis of samples previously characterised with larger AIM sets.     

Revision of the SNPforID 34-plex forensic ancestry test: Assay enhancements,standard reference sample genotypes and extended population studies

A revision of an established 34 SNP forensic ancestry test has been made by swapping the under-performing rs727811 component SNP with the highly informative rs3827760 that shows a near-fixed East Asian specific allele. We collated SNP variability data for the revised SNP set in 66 reference populations from 1000 Genomes and HGDP-CEPH panels and used this as reference data to analyse four U.S. populations showing a range of admixture patterns. The U.S. Hispanics sample in particular displayed heterogeneous values of co-ancestry between European, Native American and African contributors, likely to reflect in part, the way this disparate group is defined using cultural as well as population genetic parameters. The genotyping of over 700 U.S. population samples also provided the opportunity to thoroughly gauge peak mobility variation and peak height ratios observed from routine use of the single base extension chemistry of the 34-plex test. Finally, the genotyping of the widely used DNA profiling Standard Reference Material samples plus other control DNAs completes the audit of the 34-plex assay to allow forensic practitioners to apply this test more readily in their own laboratories.     

Analysis of the SNPforID 52-plex markers in four Native American populations from Venezuela

The SNPforID 52-plex single nucleotide polymorphisms (SNPs) were analyzed in four native Venezuelan populations: Bari, Pemon, Panare and Warao. None of the population-locus combinations showed significant departure from Hardy-Weinberg equilibrium. Calculation of forensic and statistical parameters showed lower values of genetic diversity in comparison with African and European populations, as well as other, admixed populations of neighboring regions of Caribbean, Central and South America. Significant levels of divergence were observed between the four Native Venezuelan populations as well as with other previously studied populations. Analysis of the 52-plex SNP loci with Structure provided an optimum number of population clusters of three, corresponding to Africans, Europeans and Native Americans. Analysis of admixed populations indicated a range of membership proportions for ancestral populations consisting of Native American, African and European components. The genetic differences observed in the Native American groups suggested by the 52 SNPs typed in our study are in agreement with current knowledge of the demographic history of the Americas.     

Eurasiaplex: A forensic SNP assay for differentiating European and South Asian ancestries

We have selected a set of single nucleotide polymorphisms (SNPs) with the specific aim of differentiating European and South Asian ancestries. The SNPs were combined into a 23-plex SNaPshot primer extension assay: Eurasiaplex, designed to complement an existing 34-plex forensic ancestry test with both marker sets occupying well-spaced genomic positions, enabling their combination as single profile submissions to the Bayesian Snipper forensic ancestry inference system. We analyzed the ability of Eurasiaplex plus 34plex SNPs to assign ancestry to a total 1648 profiles from 16 European, 7 Middle East, 13 Central-South Asian and 21 East Asian populations. Ancestry assignment likelihoods were estimated from Snipper using training sets of five-group data (three Eurasian groups, East Asian and African genotypes) and four-group data (Middle East genotypes removed). Five-group differentiations gave assignment success of 91% for NW European populations, 72% for Middle East populations and 39% for Central-South Asian populations, indicating Middle East individuals are not reliably differentiated from either Europeans or Central-South Asians. Four-group differentiations provided markedly improved assignment success rates of 97% for most continental Europeans tested (excluding Turkish and Adygei at the far eastern edge of Europe) and 95% for Central-South Asians, despite applying a probability threshold for the highest likelihood ratio above ‘100 times more likely’. As part of the assessment of the sensitivity of Eurasiaplex to analyze challenging forensic material we detail Eurasiaplex and 34-plex SNP typing to infer ancestry of a cranium recovered from the sea, achieving 82% SNP genotype completeness. Therefore, Eurasiaplex provides an informative and forensically robust approach to the differentiation of European and South Asian ancestries amongst Eurasian populations.     

Predicting biogeographical ancestry in admixed individuals – values and limitations of using uniparental and autosomal markers

When microsatellite profiles generated from crime scene samples do not match a known person, or eye-witness information is unreliable, highly informative uniparental and autosomal markers can help unveil biogeographical ancestry. However, as genetic admixture is becoming increasingly common in cosmopolitan societies, concern arises with their accuracy and suitability when dealing with samples from admixed individuals. Here we assess the ability to detect biogeographical ancestry in 85 individuals from self-declared Asian and European admixed families using a set of uniparental (Y and mitochondrial DNA) and autosomal single nucleotide polymorphisms, specifically selected to distinguish between these two biogeographical ancestries. Haplogroups and autosomal genotypes were investigated using STRUCTURE to detect levels of admixture. All haplogroups were characteristic of self-declared populations of origin. Overall, the autosomal markers inferred biogeographical ancestry more accurately in admixed individuals, showing no significant differences between observed and expected contribution from each population studied according to level of admixture, although some outliers were observed. We suggest a panel of highly informative autosomal and uniparental markers should be employed to infer biogeographical ancestry of an individual to help detect admixed ancestries.     

A panel of 130 autosomal single-nucleotide polymorphisms for ancestry assignment in five Asian populations and in Caucasians

Ancestry informative single-nucleotide polymorphism (AISNP) panels for differentiating between East and Southeast Asian populations are scarce. This study aimed to identify AISNPs for ancestry assignment of five East and Southeast Asian populations, and Caucasians. We analyzed 145 autosomal SNPs of the 627 DNA samples from individuals of six populations (234 Taiwanese Han, 91 Filipinos, 79 Indonesians, 60 Thais, 71 Vietnamese, and 92 Caucasians) using arrays. The multiple logistic regression model and a multi-tier approach were used for ancestry classification. We observed that 130 AISNPs were effective for classifying the ethnic origins with fair accuracy. Among the 130 AISNPs, 122 were useful for stratification between these five Asian populations and 64 were effective for differentiating between Caucasians and these Asian populations. For differentiation between Caucasians and Asians, an accuracy rate of 100% was achieved in these 627 subjects with 50 optimal AISNPs among the 64 effective SNPs. For classification of the five Asian populations, the accuracy rates of ancestry inference using 20 to 57 SNPs for each of the two Asian populations ranged from 74.1% to 100%. Another 14 degraded DNA samples with incomplete profiling were analyzed, and the ancestry of 12 (85.7%) of those subjects was accurately assigned. We developed a 130-AISNP panel for ethnic origin differentiation between the five East and Southeast Asian populations and Caucasians. This AISNP set may be helpful for individual ancestral assignment of these populations in forensic casework.     

Establishing a second-tier panel of 18 ancestry informative markers to improve ancestry distinctions among Asian populations

At present, several mature ancestry informative SNP (AISNP) panels are used to distinguish between continental regions of the world, but a more accurate division within the continent requires a secondary panel to complete. However, many AISNPs for the subgroup ancestry inference are selected from the Kidd Lab panel of 55 AISNPs or other published papers. These panels inevitably lack valuable markers for subgroup ancestry inference. Therefore, instead of choosing from the published panels, we used the 1000 Genomes Project to screen potentially informational markers in Asian populations, including single nucleotide polymorphisms (SNPs) and insertion/deletion polymorphisms (InDels). The allele frequencies of all autosomal SNPs and InDels of the 1000 Genomes Project were compared between 10 populations in Asia to identify markers with the largest pairwise allele frequency differences. Finally, we established a second-tier panel of 18 AIMs in this study, which not only divided the 26 populations of the 1000 Genomes Project into six clusters, but also divided the Asia subgroup into four clusters: Gujarati, East Asia, Southeast Asia and South Asia.     

Performance of ancestry-informative SNP and microhaplotype markers

The use of microhaplotypes (MHs) for ancestry inference has added to an increasing number of ancestry-informative markers (AIMs) for forensic application that includes autosomal single nucleotide polymorphisms (SNPs) and insertions/deletions (indels). This study compares bi-allelic and tri-allelic SNPs as well as MH markers for their ability to differentiate African, European, South Asian, East Asian, and American population groups from the 1000 Genomes Phase 3 database. A range of well-established metrics were applied to rank each marker according to the population differentiation potential they measured. These comprised: absolute allele frequency differences (δ); Rosenberg’s informativeness for (ancestry) assignment (I_n); the fixation index (F_ST); and the effective number of alleles (A_e). A panel consisting of all three marker types resulted in the lowest mean divergence per population per individual (MDPI = 2.16%) when selected by I_n. However, when marker types were not mixed, MHs were the highest performing markers by most metrics (MDPI < 4%) for differentiation between the five continental populations.     

Male lineage strata of Brazilian population disclosed by the simultaneous analysis of STRs and SNPs

Brazil has a large territory divided in five geographical regions harboring highly diverse populations that resulted from different degrees and modes of admixture between Native Americans, Europeans and Africans. In this study, a sample of 605 unrelated males was genotyped for 17 Y-STRs and 46 Y-SNPs aiming a deep characterization of the male gene pool of Rio de Janeiro and its comparison with other Brazilian populations. High values of Y-STR haplotype diversity (0.9999 ± 0.0001) and Y-SNP haplogroup diversity (0.7589 ± 0.0171) were observed. Population comparisons at both haplotype and haplogroup levels showed significant differences between Brazilian South Eastern and Northern populations that can be explained by differences in the proportion of African and Native American Y chromosomes. Statistical significant differences between admixed urban samples from the five regions of Brazil were not previously detected at haplotype level based on smaller size samples from South East, which emphasizes the importance of sample size to detected population stratification for an accurate interpretation of profile matches in kinship and forensic casework. Although not having an intra-population discrimination power as high as the Y-STRs, the Y-SNPs are more powerful to disclose differences in admixed populations. In this study, the combined analysis of these two types of markers proved to be a good strategy to predict population sub-structure, which should be taken into account when delineating forensic database strategies for Y chromosome haplotypes.     

Completion of a worldwide reference panel of samples for an ancestry informative Indel assay

The use of ancestry informative markers (AIMs) in forensic analysis is of considerable utility since ancestry inference can progress an investigation when no identification has been made of DNA from the crime-scene. Short-amplicon markers, including insertion deletion polymorphisms, are particularly useful in forensic analysis due to their mutational stability, capacity to amplify degraded samples and straightforward amplification technique. In this study we report the completion of H952 HGDP–CEPH panel genotyping with a set of 46 AIM-Indels. The study adds Central South Asian and Middle Eastern population data, allowing a comparison of patterns of variation in Eurasia for these markers, in order to enhance their use in forensic analyses, particularly when combined with sets of ancestry informative SNPs. Ancestry analysis using principal component analysis and Bayesian methods indicates that a proportion of classification error occurs with European–Middle East population comparisons, but the 46 AIM-Indels have the capability to differentiate six major population groups when European–Central South Asian comparisons are made. These findings have relevance for forensic ancestry analyses in countries where South Asians form much of the demographic profile, including the UK, USA and South Africa. A novel third allele detected in MID-548 was characterized – despite a low frequency in the HGDP–CEPH panel samples, it appears confined to Central South Asian populations, increasing the ability to differentiate this population group. The H952 data set was implemented in a new open access SPSmart frequency browser – forInDel: Forensic Indel browser.     

Application of a custom haplotype caller to analyze sequence-based data of 56 microhaplotypes

Microhaplotypes (microhaps) are recently introduced markers that aim to complement the limitations of conventional forensic markers such as short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). With the potential of microhaps in forensics becoming clearer through massively parallel sequencing (MPS), MPS-based studies on microhaps are being actively reported. However, simpler workflow schemes for the generation and analysis of MPS data are still required to facilitate the practical application of MPS in forensics. In this study, we developed an in-house MPS panel that simultaneously amplifies 56 microhaps and a custom haplotype caller, Visual Microhap. The developed tool works on a web browser and provides four analysis options to extract SNP-based haplotypes from sequence-based data obtained by STRait Razor 3.0. To demonstrate the utility of the MPS panel and data analysis workflow scheme, we also analyzed 56 microhaps of 286 samples from four populations (African-American, Caucasian, Hispanic, and Korean). The average effective number of alleles (A_e) for the four groups was 3.45, ranging from 1.74 to 6.98. Forensic statistical parameters showed that this microhap panel is more powerful than conventional autosomal STRs for human identification. Meanwhile, the 56-plex panel mostly comprised microhaps with high Ae; however, the four populations were grossly distinguishable from each other by cluster analysis. Consequently, the developed in-house MPS panel for 56 microhaps and the adopted workflow using open-source tools can increase the utility of microhap MPS in forensic research and practice.     

Eurasiaplex-2: Shifting the focus to SNPs with high population specificity increases the power of forensic ancestry marker sets

To compile a new South Asian-informative panel of forensic ancestry SNPs, we changed the strategy for selecting the most powerful markers for this purpose by targeting polymorphisms with near absolute specificity – when the South Asian-informative allele identified is absent from all other populations or present at frequencies below 0.001 (one in a thousand). More than 120 candidate SNPs were identified from 1000 Genomes datasets satisfying an allele frequency screen of ≥ 0.1 (10 % or more) allele frequency in South Asians, and ≤ 0.001 (0.1 % or less) in African, East Asian, and European populations. From the candidate pool of markers, a final panel of 36 SNPs, widely distributed across most autosomes, were selected that had allele frequencies in the five 1000 Genomes South Asian populations ranging from 0.4 to 0.15. Slightly lower average allele frequencies, but consistent patterns of informativeness were observed in gnomAD South Asian datasets used to validate the 1000 Genomes variant annotations. We named the panel of 36 South Asian-specific SNPs Eurasiaplex-2, and the informativeness of the panel was evaluated by compiling worldwide population data from 4097 samples in four genome variation databases that largely complement the global sampling of 1000 Genomes. Consistent patterns of allele frequency distribution, which were specific to South Asia, were observed in all populations in, or closely sited to, the Indian sub-continent. Pakistani populations from the HGDP-CEPH panel had markedly lower allele frequencies, highlighting the need to develop a statistical system to evaluate the ancestry inference value of counting the number of population-specific alleles present in an individual.     

Pacifiplex: an ancestry-informative SNP panel centred on Australia and the Pacific region

The analysis of human population variation is an area of considerable interest in the forensic, medical genetics and anthropological fields. Several forensic single nucleotide polymorphism (SNP) assays provide ancestry-informative genotypes in sensitive tests designed to work with limited DNA samples, including a 34-SNP multiplex differentiating African, European and East Asian ancestries. Although assays capable of differentiating Oceanian ancestry at a global scale have become available, this study describes markers compiled specifically for differentiation of Oceanian populations. A sensitive multiplex assay, termed Pacifiplex, was developed and optimized in a small-scale test applicable to forensic analyses. The Pacifiplex assay comprises 29 ancestry-informative marker SNPs (AIM-SNPs) selected to complement the 34-plex test, that in a combined set distinguish Africans, Europeans, East Asians and Oceanians. Nine Pacific region study populations were genotyped with both SNP assays, then compared to four reference population groups from the HGDP-CEPH human diversity panel. STRUCTURE analyses estimated population cluster membership proportions that aligned with the patterns of variation suggested for each study population’s currently inferred demographic histories. Aboriginal Taiwanese and Philippine samples indicated high East Asian ancestry components, Papua New Guinean and Aboriginal Australians samples were predominantly Oceanian, while other populations displayed cluster patterns explained by the distribution of divergence amongst Melanesians, Polynesians and Micronesians. Genotype data from Pacifiplex and 34-plex tests is particularly well suited to analysis of Australian Aboriginal populations and when combined with Y and mitochondrial DNA variation will provide a powerful set of markers for ancestry inference applied to modern Australian demographic profiles. On a broader geographic scale, Pacifiplex adds highly informative data for inferring the ancestry of individuals from Oceanian populations. The sensitivity of Pacifiplex enabled successful genotyping of population samples from 50-year-old serum samples obtained from several Oceanian regions that would otherwise be unlikely to produce useful population data. This indicates tests primarily developed for forensic ancestry analysis also provide an important contribution to studies of populations where useful samples are in limited supply.     

Indel markers: genetic diversity of 38 polymorphisms in Brazilian populations and application in a paternity investigation with post mortem material

Aiming to evaluate the usefulness of 38 non-coding bi-allelic autosomal indels in genetic identification and kinship testing, three Brazilian population samples were studied: two from Rio de Janeiro (including a sample of individuals with self-declared African ancestry) and one Native American population of Terena from Mato Grosso do Sul. Based on the observed allele frequencies, parameters of forensic relevance were calculated. The combined power of discrimination of the 38 indels was high in all studied groups (PD≥0.9999999999997), although slightly lower in Native Americans. Genetic distance analysis showed significant differences between the allele frequencies in the Rio de Janeiro population and those previously reported for Europeans, Africans and Asians explained by its intermediate position between Europeans and Africans. As expected, the Terena sample was significantly different from all the other populations: Brazilians from Rio de Janeiro general population and with self-declared African ancestry, Europeans, Africans and East Asians. Finally, the performance of the 38-indel multiplex assay was tested in post-mortem material with positive results, supporting the use of short amplicon bi-allelic markers as an additional tool to STR analysis when DNA molecules are degraded.