Ancestry informative markers: Inference of ancestry in aged bone samples using an autosomal AIM-Indel multiplex

Ancestry informative markers (AIMs) can be useful to infer ancestry proportions of the donors of forensic evidence. The probability of success typing degraded samples, such as human skeletal remains, is strongly influenced by the DNA fragment lengths that can be amplified and the presence of PCR inhibitors. Several AIM panels are available amongst the many forensic marker sets developed for genotyping degraded DNA. Using a 46 AIM Insertion Deletion (Indel) multiplex, we analyzed human skeletal remains of post mortem time ranging from 35 to 60 years from four different continents (Sub-Saharan Africa, South and Central America, East Asia and Europe) to ascertain the genetic ancestry components. Samples belonging to non-admixed individuals could be assigned to their corresponding continental group. For the remaining samples with admixed ancestry, it was possible to estimate the proportion of co-ancestry components from the four reference population groups. The 46 AIM Indel set was informative enough to efficiently estimate the proportion of ancestry even in samples yielding partial profiles, a frequent occurrence when analyzing inhibited and/or degraded DNA extracts.     

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.     

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.     

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.     

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.     

Inferring ancestral origin using a single multiplex assay of ancestry-informative marker SNPs

Tests that infer the ancestral origin of a DNA sample have considerable potential in the development of forensic tools that can help to guide crime investigation. We have developed a single-tube 34-plex SNP assay for the assignment of ancestral origin by choosing ancestry-informative markers (AIMs) exhibiting highly contrasting allele frequency distributions between the three major population-groups. To predict ancestral origin from the profiles obtained, a classification algorithm was developed based on maximum likelihood. Sampling of two populations each from African, European and East Asian groups provided training sets for the algorithm and this was tested using the CEPH Human Genome Diversity Panel. We detected negligible theoretical and practical error for assignments to one of the three groups analyzed with consistently high classification probabilities, even when using reduced subsets of SNPs. This study shows that by choosing SNPs exhibiting marked allele frequency differences between population-groups a practical forensic test for assigning the most likely ancestry can be achieved from a single multiplexed assay.     

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.     

Analysis of the South Australian Aboriginal population using the Global AIMs Nano ancestry test

We investigate the ability of the 31 SNP loci in the Global AIMs Nano set to distinguish self-declared Australian Aboriginal individuals from European, Oceanic, African, Native American and East Asian populations. Human evolution suggests that Australian Aboriginal individuals came to Australia approximately 50 000 years ago, during the time it made up part of Sahul. Since then the colonisation of Australia by Europeans has meant significant admixture within the Australian Aboriginal population. These two events present themselves in our study with the Aboriginal population creating a continuous genetic cline between the Oceanic and European groups. We also assigned the Aboriginal individuals into their traditional regional groups to determine whether there was any ability to distinguish these from each other. We found similar results to studies using other markers, namely that the more remote regions (that have been less affected by admixture) diverged from the rest. Overall, we found the ability of the GNano system to differentiate self-declared Australian Aboriginal individuals was reasonable but had limitations that need to be recognised if these assignments are applied to unknown individuals.     

Exploring of new Y-chromosome SNP loci using Pyrosequencing and the SNaPshot methods

The single nucleotide polymorphisms on the Y chromosome (Y-SNP) have been considered to be important in forensic casework. However, Y-SNP loci were mostly population specific and lacked biallelic polymorphisms in the Asian population. In this study, we developed a strategy for seeking and genotyping new Y-SNP markers based on both Pyrosequencing and the SNaPshot methods. As results, 34 new biallelic markers were observed to be polymorphic in the Chinese Han population by estimation of allele frequencies of 103 candidate’s Y-SNP loci in DNA pools using Pyrosequencing technology. Then, a multiplex system with 20 Y-SNP loci was genotyped using the SNaPshot? multiplex kit. Twenty Y-SNP loci defined 56 different haplotypes, and the haplotype diversity was estimated to be 0.9539. Our result demonstrated that the strategy could be used as an efficient tool to search and genotype biallelic markers from a large amount of candidate loci. In addition, 20 Y-SNP loci constructed a multiplex system, which could provide supplementary information for forensic identification.     

A 21 marker insertion deletion polymorphism panel to study biogeographic ancestry

Insertion/deletion polymorphisms have recently received increased interest in the forensic genetics community. This class of markers combines the advantageous genetic properties of single nucleotide polymorphisms (i.e., low mutation rate, genetic stability, and short amplicon size) with the technical advantage of short tandem repeat markers (simple detection by fluorescence-labelled PCR and capillary electrophoresis). For a large number of indel markers significant differences in allele frequencies between the major populations have been reported, making this class of markers suitable for the analysis of biogeographic ancestry. We have developed a multiplex PCR assay designed to establish the biogeographic ancestry of forensic DNA samples based on insertion/deletion polymorphisms. A panel of 21 short indels with allele frequency differences between three major population groups (European, African and Asian) was selected to be incorporated into a single-tube multiplex PCR assay. The assay is highly sensitive, requiring less than 0.5 ng of genomic DNA for successful typing. Due to the short fragment lengths below 200 bp, the assay is ideally suited for the typing of challenging forensic genetic case work samples. A population genetic study has been performed proving the performance of the assay in inferring the ancestral population of individuals. The chosen 21 markers are sufficient to distinguish between three major global population groups. Furthermore, the assay design leaves room for an extension in order to cover additional population groups.     

Identification and characterization of novel DIP-STRs from whole-genome sequencing data

In an attempt to enhance forensic DNA mixture deconvolution several alternative DNA typing approaches have been developed. Among these, DIP-STR compound markers can resolve extremely unbalanced two-source DNA mixtures of same-or-opposite sex donors, up to a 1:1000 minor:major DNA ratio. A forensic set of 10 markers was validated for casework and a larger set of 23 DIP-STRs has proven suitable to biogeographic ancestry inference and for prenatal paternity testing. Yet, to promote the widespread use of this original approach, more markers and multiplex panels need to be developed.To this end, here we describe an extended set of forensic DIP-STRs identified using currently available whole-genome sequencing datasets. Complete lists of Indels and STRs were obtained from reported frequencies of genetic variants of 76,156 genomes. About 3000 identified DIP-STRs candidates were shorter than 200 bp and 500 showed high haplotype variability estimated using the genotypes of individuals homozygous for the DIP or the STR. Here, we present 23 additional DIP-STRs validated for sensitivity, specificity and Swiss population variability. Finally, a set of 30 markers comprising seven previously validated ones is proposed for the prospective development of a forensic DIP-STR multiplex panel.     

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory’s data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.     

Epigenome-wide screening of CpG markers to develop a multiplex methylation SNaPshot assay for age prediction

Age prediction can provide important information about the contributors of biological evidence left at crime scenes. DNA methylation has been regarded as the most promising age-predictive biomarker. Measuring the methylation level at the genome-wide scale is an important step to screen specific markers for forensic age prediction. In present study, we screened out five age-related CpG sites from the public EPIC BeadChip data and evaluated them in a training set (115 blood) by multiplex methylation SNaPshot assay. Through full subset regression, the five markers were narrowed down to three, namely cg10501210 (C1orf132), cg16867657 (ELOVL2), and cg13108341 (DNAH9), of which the last one was a newly discovered age-related CpG site. An age prediction model was built based on these three markers, explaining 86.8% of the variation of age with a mean absolute deviation (MAD) of 4.038 years. Then, the multiplex methylation SNaPshot assay was adjusted according to the age prediction model. Considering that bloodstains are one of the most common biological samples in practical cases, three validation sets composed of 30 blood, 30 fresh bloodstains and 30 aged bloodstains were used for evaluation of the age prediction model. The MAD of each set was estimated as 4.734, 4.490, and 5.431 years, respectively, suggesting that our age prediction model was applicable for age prediction for blood and bloodstains in Chinese Han population of 11–71 age. In general, this study describes a workflow of screening CpG markers from public chip data and presents a 3-CpG markers model for forensic age prediction.     

Evaluation of 12 Multi-InDel markers for forensic ancestry prediction in Asian populations

Various types of genetic markers have been applied to forensic ancestry inference. Biallelic markers, such as SNPs and InDels, have proven to be optimal choices except for the low information content provided by a single locus. Multi-InDel marker is defined as a specific DNA fragment with several InDel markers located tightly in the physical position. Previous research indicates that multi-InDel markers perform well in population analysis and ancestry inference because of higher degree of polymorphism and remarkable population differences. In this study, a panel consisting of 12 multi-InDel markers was employed to evaluate the general performance in forensic practice and the discrimination power for population analysis. Sample types encountered in routine forensic practice were genotyped to validate the feasibility of regular use. A population study was performed on a total of five Asian populations to verify the discrimination power. Moreover, a double-blind test for ancestry prediction was conducted to assess the predictive capability. In conclusion, these results revealed the significance of multi-InDel markers for population structure stratification. The present panel showed the potential as a valid complementary tool in forensic applications.     

MAPlex - A massively parallel sequencing ancestry analysis multiplex for Asia-Pacific populations

Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.     

Design and validation of a highly discriminatory 10-locus Y-chromosome STR multiplex system

The Y-chromosome STRs (short tandem repeat) markers are routinely utilized in the resolution of forensic casework related to sexual assault. For this, the forensic community has adopted a set of eleven (core) Y-STR that is incorporated in all commercial diagnostic systems. Our previous studies of Y-STR polymorphisms in the South African population identified low levels of diversity and discrimination capacity for many commercial marker sets, determining a limited applicability of these systems to the local population groups. To overcome this shortcoming, we designed a Y-STR 10-plex system that shows higher discriminatory capacity (DC) than available commercial systems. The markers were selected from a population group of 283 individuals with African, European and Asian ancestry genotyped at 45 Y-STRs, applying an optimization based selection procedure to achieve the highest possible DC with the minimal number of markers. The 10-plex was satisfactorily subjected to developmental validation tests following the SWGDAM guidelines and shows potential for its application to genealogical and evolutionary studies.     

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.     

Development of a SNP set for human identification: A set with high powers of discrimination which yields high genetic information from naturally degraded DNA samples in the Thai population

This study describes the development of a SNP typing system for human identification in the Thai population, in particular for extremely degraded DNA samples. A highly informative SNP marker set for forensic identification was identified, and a multiplex PCR-based Invader assay was developed. Fifty-one highly informative autosomal SNP markers and three sex determination SNP markers were amplified in two multiplex PCR reactions and then detected using Invader assay reactions. The average PCR product size was 71 base pairs. The match probability of the 54-SNP marker set in 124 Thai individuals was 1.48 × 10⁻²¹, higher than that of STR typing, suggesting that this 54-SNP marker set is beneficial for forensic identification in the Thai population. The selected SNP marker set was also evaluated in 90 artificially degraded samples, and in 128 naturally degraded DNA samples from real forensic casework which had shown no profiles or incomplete profiles when examined using a commercial STR typing system. A total of 56 degraded samples (44%) achieved the matching probability (PM) equivalent to STR gold standard analysis (successful genotyping of 44 SNP markers) for human identification. These data indicated that our novel 54-SNP marker set provides a very useful and valuable approach for forensic identification in the Thai population, especially in the case of highly to extremely degraded DNA.In summary, we have developed a set of 54 Thai-specific SNPs for human identification which have higher discrimination power than STR genotyping. The PCRs for these 54 SNP markers were successfully combined into two multiplex reactions and detected with an Invader assay. This novel SNP genotyping system also yields high levels of genetic information from naturally degraded samples, even though there are much more difficult to recover than artificially degraded samples.