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.     

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.     

AIM-SNPtag: A computationally efficient approach for developing ancestry-informative SNP panels

Inferring an individual's ancestry or group membership using a small set of highly informative genetic markers is very useful in forensic and medical genetics. However, given the huge amount of SNP data available from a diverse of populations, it is challenging to develop informative panels by exhaustively searching for all possible SNP combinations. In this study, we formulate it as an algorithm problem of selecting an optimal set of SNPs that maximizes the inference accuracy while minimizes the set size. Built on this conception, we develop a computational approach that is capable of constructing ancestry informative panels from multi-population genome-wide SNP data efficiently. We evaluated the performance of the method by comparing the panel size and membership inference accuracy of the constructed SNP panels to panels selected through empirical procedures in previous studies. For the membership inference of population groups including Asian, European, African, East Asian and Southeast Asian, a 36-SNP panel developed by our approach has an overall accuracy of 99.07%, and a 21-SNP subset of the panel has an overall accuracy of 95.36%. In comparison, an existing panel requires 74 SNPs to achieve an accuracy of 94.14% on the same set of population groups. We further apply the method to four subpopulations within Europe (Finnish, British, Spanish and Italian); a 175-SNP panel can discriminate individuals of those European subpopulations with an accuracy of 99.36%, of which a 68-SNP subset can achieve an accuracy of 95.07%. We expect our method to be a useful tool for constructing ancestry informative markers in forensic genetics.     

Biogeographical ancestry,variable selection,and PLS-DA method: a new panel to assess ancestry in forensic samples via MPS technology

As evidenced by the large number of articles recently published in the literature, forensic scientists are making great efforts to infer externally visible features and biogeographical ancestry (BGA) from DNA analysis. Just as phenotypic, ancestry information obtained from DNA can provide investigative leads to identify the victims (missing/unidentified persons, crime/armed conflict/mass disaster victims) or trace their perpetrators when no matches were found with the reference profile or in the database. Recently, the advent of Massively Parallel Sequencing technologies associated with the possibility of harnessing high-throughput genetic data allowed us to investigate the associations between phenotypic and genomic variations in worldwide human populations and develop new BGA forensic tools capable of simultaneously analyzing up to millions of markers if for example the ancient DNA approach of hybridization capture was adopted to target SNPs of interest. In the present study, a selection of more than 3000 SNPs was performed to create a new BGA panel and the accuracy of the new panel to infer ancestry from unknown samples was evaluated by the PLS-DA method. Subsequently, the panel created was assessed using three variable selection techniques (Backward variable elimination, Genetic Algorithm and Regularized elimination procedure), and the best SNPs in terms of inferring bio-geographical ancestry at inter- and intra-continental level were selected to obtain panels to predict BGA with a reduced number of selected markers to be applied in routine forensic cases where PCR amplification is the best choice to target SNPs.     

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.     

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.     

The frequency of fingerprint patterns separated by ethnicity and sex in a general population from Sydney,Australia

ABSTRACT

A total of 514 individuals from the Sydney area volunteered their fingerprint scans for analysis. The prints were classified using the eight main patterns of the National Crime Information Centre system. Additional information from the volunteers, including sex and ancestry, was documented. Using this information, the frequencies of each pattern for each biogeographical ancestry (BGA) group and sex were calculated. Trends showed that males and females have a difference in the frequency of plain arches; none of the other patterns showed clear differentiation. BGA trends showed that European and Middle Eastern groups had more similar frequencies for the less common patterns (plain arch, tented arch, radial loop, central pocket loop whorl and double loop whorl). Whereas for the common patterns of ulnar loop and plain whorl, European and South Asian groups were more similar in frequency.     

Application of machine learning for ancestry inference using multi-InDel markers

Ancestry inference through population stratification plays an important role in forensic applications. Specifically, ancestry information inferred from forensic DNA evidence can provide vital clues for criminal investigations. Current advances in ancestry inference mostly focus on ancestry informative markers. Hereinto, multi-InDel was proposed as one of the compound markers performing well in complex ancestral classification in the subpopulation of Asia. However, research on analytical methods necessary to make reliable predictions is lacking. The newly proposed compound markers could be assessed with alternative methods. In this study, promising discriminant methods were explored using multi-InDel markers for forensic ancestry inference. As a prerequisite, the adopted multi-InDel markers were assessed by classical methods for population genetics, such as F_ST analysis, MDS and STRUCTURE. In addition, dimensionality reduction methods and serial reduction strategies were applied for data visualization. Subsequently, machine learning methods, including logistic regression (LR), support vector machine (SVM), k-nearest neighbors (KNN) and extreme gradient boosting (XGBoost), were evaluated by diverse approaches. As the result of multifarious analyses through comparisons and estimations, XGBoost with one-hot encoding was shown to be more effective in population stratification and ancestry inference for challenging cases with admixed populations.     

Inference of recent admixture using genotype data

The inference of biogeographic ancestry (BGA) has become a focus of forensic genetics. Misinference of BGA can have profound unwanted consequences for investigations and society. We show that recent admixture can lead to misclassification and erroneous inference of ancestry proportions, using state of the art analysis tools with (i) simulations, (ii) 1000 genomes project data, and (iii) two individuals analyzed using the ForenSeq DNA Signature Prep Kit. Subsequently, we extend existing tools for estimation of individual ancestry (IA) by allowing for different IA in both parents, leading to estimates of parental individual ancestry (PIA), and a statistical test for recent admixture. Estimation of PIA outperforms IA in most scenarios of recent admixture. Furthermore, additional information about parental ancestry can be acquired with PIA that may guide casework.     

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.     

Development and evaluations of the ancestry informative markers of the VISAGE Enhanced Tool for Appearance and Ancestry

The VISAGE Enhanced Tool for Appearance and Ancestry (ET) has been designed to combine markers for the prediction of bio-geographical ancestry plus a range of externally visible characteristics into a single massively parallel sequencing (MPS) assay. We describe the development of the ancestry panel markers used in ET, and the enhanced analyses they provide compared to previous MPS-based forensic ancestry assays. As well as established autosomal single nucleotide polymorphisms (SNPs) that differentiate sub-Saharan African, European, East Asian, South Asian, Native American, and Oceanian populations, ET includes autosomal SNPs able to efficiently differentiate populations from Middle East regions. The ability of the ET autosomal ancestry SNPs to distinguish Middle East populations from other continentally defined population groups is such that characteristic patterns for this region can be discerned in genetic cluster analysis using STRUCTURE. Joint cluster membership estimates showing individual co-ancestry that signals North African or East African origins were detected, or cluster patterns were seen that indicate origins from central and Eastern regions of the Middle East. In addition to an augmented panel of autosomal SNPs, ET includes panels of 85 Y-SNPs, 16 X-SNPs and 21 autosomal Microhaplotypes. The Y- and X-SNPs provide a distinct method for obtaining extra detail about co-ancestry patterns identified in males with admixed backgrounds. This study used the 1000 Genomes admixed African and admixed American sample sets to fully explore these enhancements to the analysis of individual co-ancestry. Samples from urban and rural Brazil with contrasting distributions of African, European, and Native American co-ancestry were also studied to gauge the efficiency of combining Y- and X-SNP data for this purpose. The small panel of Microhaplotypes incorporated in ET were selected because they showed the highest levels of haplotype diversity amongst the seven population groups we sought to differentiate. Microhaplotype data was not formally combined with single-site SNP genotypes to analyse ancestry. However, the haplotype sequence reads obtained with ET from these loci creates an effective system for de-convoluting two-contributor mixed DNA. We made simple mixture experiments to demonstrate that when the contributors have different ancestries and the mixture ratios are imbalanced (i.e., not 1:1 mixtures) the ET Microhaplotype panel is an informative system to infer ancestry when this differs between the contributors.     

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.     

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.     

Off-target phenotypes in forensic DNA phenotyping and biogeographic ancestry inference: A resource

With recent advances in DNA sequencing technologies it has become feasible and cost effective to genotype larger marker sets for forensic purposes. Two technologies that make use of the larger marker sets have come into focus in forensic research and applications; inference of biogeographic ancestry (BGA) and forensic DNA phenotyping (FDP). These methods hold the promise to reveal information about a yet unknown perpetrator from a DNA sample. In contrast, DNA-profiling, that is a standard practice in case work, relies on matching DNA-profiles between crime scene material and suspects on a database of DNA-profiles. Markers for DNA-profiling were developed under the premise to reveal as little additional information about the human source of the profile as possible, the rationale being that personal privacy rights have to be balanced against the public interest in solving a crime. The same argument holds for markers used in BGA and FDP; these markers might also reveal information on off-target phenotypes (OTPs), that go beyond BGA and the phenotypes targeted in FDP. In particular, health related OTPs might shift the balance between privacy protection and public interest. However, to our knowledge, there is currently no convenient resource available to incorporate knowledge on OTPs in BGA and FDP assay design and application.In order to provide such a resource, we performed a systematic search for OTPs associated with a comprehensive set of markers (1766 SNPs) used or suggested to be used for BGA inference and FDP. In this set, we identified a relatively small number of 27 SNPs (1.53%) that convey information on diverse health related OTPs such as cancer risk, induced asthma, or risk of alcoholism. Some of these SNPs are commonly used for FDP and BGA across different marker sets. We conclude that the effects of SNP markers used in FDP and BGA on OTPs are currently limited, with few exceptions that should be considered in a balanced decision on assay design and application.     

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 SNaPshot of next generation sequencing for forensic SNP analysis

Forensic phenotyping can provide useful intelligence regarding the biogeographical ancestry (BGA) and externally visible characteristics (EVCs) of the donor of an evidentiary sample. Currently, single nucleotide polymorphism (SNP) based inference of BGA and EVCs is performed most commonly using SNaPshot^®, a single base extension (SBE) assay. However, a single SNaPshot multiplex PCR is limited to 30–40 SNPs. Next generation sequencing (NGS) offers the potential to genotype hundreds to thousands of SNPs from multiple samples in a single experimental run. The PCR multiplexes from five SNaPshot assays (SNPforID 52plex, SNPforID 34plex, Eurasiaplex, IrisPlex and an unpublished BGA assay) were applied to three different DNA template amounts (0.1, 0.2 and 0.3 ng) in three samples (9947A and 007 control DNAs and a male donor). The pooled PCR amplicons containing 136 unique SNPs were sequenced using Life Technologies’ Ion Torrent™ PGM system. Approximately 72 Mb of sequence was generated from two 10 Mb Ion 314™ v1 chips. Accurate genotypes were readily obtained from all three template amounts. Of a total of 408 genotypes, 395 (97%) were fully concordant with SNaPshot across all three template amounts. Of those genotypes discordant with SNaPshot, six Ion Torrent sequences (1.5%) were fully concordant with Sanger sequencing across the three template amounts. Seven SNPs (1.7%) were either discordant between template amounts or discordant with Sanger sequencing. Sequence coverage observed in the negative control, and, allele coverage variation for heterozygous genotypes highlights the need to establish a threshold for background levels of sequence output and heterozygous balance. This preliminary study of the Ion Torrent PGM system has demonstrated considerable potential for use in forensic DNA analyses as a low to medium throughput NGS platform using established SNaPshot assays.     

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.     

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.