Biogeographical Ancestry Inference Research Articles

Biogeographical Ancestry Analyses Using the ForenSeqTM DNA Signature Prep Kit and Multiple Prediction Tools.

The inference of biogeographical ancestry (BGA) can assist in police investigations of serious crime cases and help to identify missing people and victims of mass disasters. In this study, we evaluated the typing performance of 56 ancestry-informative SNPs in 177 samples using the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx system. Furthermore, we compared the prediction accuracy of the tools Universal Analysis Software v1.2 (UAS), the FROG-kb, and GenoGeographer when inferring the ancestry of 503 Europeans, 22 non-Europeans, and 5 individuals with co-ancestry. The kit was highly sensitive with complete aiSNP profiles in samples with as low as 250pg input DNA. However, in line with others, we observed low read depth and occasional drop-out in some SNPs. Therefore, we suggest not using less than the recommended 1ng of input DNA. FROG-kb and GenoGeographer accurately predicted both Europeans (99.6% and 91.8% correct, respectively) and non-Europeans (95.4% and 90.9% correct, respectively). The UAS was highly accurate when predicting Europeans (96.0% correct) but performed poorer when predicting non-Europeans (40.9% correct). None of the tools were able to correctly predict individuals with co-ancestry. Our study demonstrates that the use of multiple prediction tools will increase the prediction accuracy of BGA inference in forensic casework.

Joint application of A-InDels and miniSTRs for forensic personal, full and half sibling identifications, and genetic differentiation analyses in two populations from China

BackgroundPreviously, a novel multiplex system of 64 loci was constructed based on capillary electrophoresis platform, including 59 autosomal insertion/deletions (A-InDels), two Y-chromosome InDels, two mini short tandem repeats (miniSTRs), and an Amelogenin gene. The aim of this study is to evaluate the efficiencies of this multiplex system for individual identification, paternity testing and biogeographic ancestry inference in Chinese Hezhou Han (CHH) and Hubei Tujia (CTH) groups, providing valuable insights for forensic anthropology and population genetics research.ResultsThe cumulative values of power of discrimination (CDP) and probability of exclusion (CPE) for the 59 A-InDels and two miniSTRs were 0.99999999999999999999999999754, 0.99999905; and 0.99999999999999999999999999998, 0.99999898 in CTH and CHH groups, respectively. When the likelihood ratio thresholds were set to 1 or 10, more than 95% of the full sibling pairs could be identified from unrelated individual pairs, and the false positive rates were less than 1.2% in both CTH and CHH groups. Biogeographic ancestry inference models based on 35 populations were constructed with three algorithms: random forest, adaptive boosting and extreme gradient boosting, and then 10-fold cross-validation analyses were applied to test these three models with the average accuracies of 86.59%, 84.22% and 87.80%, respectively. In addition, we also investigated the genetic relationships between the two studied groups with 33 reference populations using population statistical methods of FST, DA, phylogenetic tree, PCA, STRUCTURE and TreeMix analyses. The present results showed that compared to other continental populations, the CTH and CHH groups had closer genetic affinities to East Asian populations.ConclusionsThis novel multiplex system has high CDP and CPE in CTH and CHH groups, which can be used as a powerful tool for individual identification and paternity testing. According to various genetic analysis methods, the genetic structures of CTH and CHH groups are relatively similar to the reference East Asian populations.

A developmental validation of the Quick TargSeq 1.0 integrated system for automated DNA genotyping in forensic science for reference samples.

Analysis of short tandem repeats (STRs) is a global standard method for human identification. Insertion/Deletion polymorphisms (DIPs) can be used for biogeographical ancestry inference. Current DNA typing involves a trained forensic worker operating several specialized instruments in a controlled laboratory environment, which takes 6-8h. We developed the Quick TargSeq 1.0 integrated system (hereinafter abbreviated to Quick TargSeq) for automated generation of STR and DIP profiles from buccal swab samples and blood stains. The system fully integrates the processes of DNA extraction, polymerase chain reaction (PCR) amplification, and electrophoresis separation using microfluidic biochip technology. Internal validation studies were performed using RTyper 21 or DIP 38 chip cartridges with single-source reference samples according to the Scientific Working Group for DNA Analysis Methods guidelines. These results indicated that the Quick TargSeq system can process reference samples and generate STR or DIP profiles in approximately 2h, and the profiles were concordant with those determined using traditional STR or DIP analysis methods. Thus, reproducible and concordant DNA profiles were obtained from reference samples. Throughout the study, no lane-to-lane or run-to-run contamination was observed. The Quick TargSeq system produced full profiles from buccal swabs with at least eight swipes, dried blood spot cards with two 2-mm disks, or 10ng of purified DNA. Potential PCR inhibitors (i.e., coffee, smoking tobacco, and chewing tobacco) did not appear to affect the amplification reactions of the instrument. The overall success rate and concordance rate of 153 samples were 94.12% and 93.44%, respectively, which is comparable to other commercially available rapid DNA instruments. A blind test initiated by a DNA expert group showed that the system can correctly produce DNA profiles with 97.29% genotype concordance with standard bench-processing methods, and the profiles can be uploaded into the national DNA database. These results demonstrated that the Quick TargSeq system can rapidly generate reliable DNA profiles in an automated manner and has the potential for use in the field and forensic laboratories.

Systematic analyses of AISNPs screening and classification algorithms based on genome-wide data for forensic biogeographic ancestry inference

Identifying the biogeographic ancestral origin of biological sample left at a crime scene can provide important evidence for judicial case, as well as clue for narrowing down suspect. Ancestry informative single nucleotide polymorphism (AISNP) has become one of the most important genetic markers in recent years for screening ancestry information loci and analyzing the population genetic background and structure due to their high number and wide distributions in the human genome. In this study, based on data from 26 populations in the 1000 Genomes Project Phase 3, a Random Forest classification model was constructed with one-vs-rest classification strategy for embedded feature selection in order to obtain a panel with a small number of efficient AISNPs. The research aim was to clarify differentiations of population genetic structures among continents and subregions of East Asia. ADMIXTURE results showed that based on the 58 AISNPs selected by the machine learning algorithm, the 26 populations involved in the study could be categorized into six intercontinental ancestry components: North East Asia, South East Asia, Africa, Europe, South Asia, and America. The 24 continental-specific AISNPs and 34 East Asian-specific AISNPs were finally obtained, and used to construct the ancestry prediction model using XGBoost algorithm, resulting in the Matthews correlation coefficients of 0.94 and 0.89, and accuracies of 0.94 and 0.92, respectively. The machine learning models that we constructed using population-specific AISNPs were able to accurately predict the ancestral origins of continental and intra-East Asian populations. To summarize, screening a set of high-perform AISNPs to infer biogeographical ancestral information using embedded feature selection has potential application in creating a layered inference system that accurately differentiates from intercontinental populations to local subpopulations.

Genetic polymorphism analysis and forensic application evaluation of 57 insertion/deletion polymorphisms from Yi ethnic group in Yunnan

Background As a new kind of diallelic genetic marker, insertion/deletion (InDel) polymorphisms have recently been used in forensic science. However, there are relatively few studies on the forensic evaluation of InDel genetic polymorphisms from different populations. Aim The aim of the present work is to assess the genetic polymorphism and forensic applicability of 57 InDels from the Yi ethnic group and explore the genetic background of this group. Subjects and methods A total sample of 122 unrelated individuals of Yi group from the Yunnan province were genotyped by the AGCU indel 60 Kit. Multiplex population genetic analyses on the same 57 InDels were carried out among the Yunnan Yi group and 29 reference populations. Results The average allele frequency of these loci in the Yi ethnic group was 0.485. Heterozygosity, polymorphism information content, and the power of discrimination were 0.477, 0.362, and 0.612, respectively. The combined power of discrimination and the combined power of exclusion reached to 0.99999999999999999669 and 0.999962965, respectively. The results showed that 57 InDels polymorphisms have high genetic polymorphisms in the Yi ethnic group. Conclusions The 57 InDels could be used for forensic individual identification, paternity testing, and intercontinental population discrimination, with the potential for use in biogeographic ancestry inference.

Comprehensive landscape of non-CODIS STRs in global populations provides new insights into challenging DNA profiles

The worldwide implementation of short tandem repeats (STR) profiles in forensic genetics necessitated establishing and expanding the CODIS core loci set to facilitated efficient data management and exchange. Currently, the mainstay CODIS STRs are adopted in most general-purpose forensic kits. However, relying solely on these loci failed to yield satisfactory results for challenging tasks, such as bio-geographical ancestry inference, complex DNA mixture profile interpretation, and distant kinship analysis. In this context, non-CODIS STRs are potent supplements to enhance the systematic discriminating power, particularly when combined with the high-throughput next-generation sequencing (NGS) technique. Nevertheless, comprehensive evaluation on non-CODIS STRs in diverse populations was scarce, hindering their further application in routine caseworks. To address this gap, we investigated genetic variations of 178 historically available non-CODIS STRs from ethnolinguistically different worldwide populations and studied their characteristics and forensic potentials via high-coverage whole genome sequencing (WGS) data. Initially, we delineated the genomic properties of these non-CODIS markers through sequence searching, repeat structure scanning, and manual inspection. Subsequent population genetics analysis suggested that these non-CODIS STRs had comparable polymorphism levels and forensic utility to CODIS STRs. Furthermore, we constructed a theoretical next-generation sequencing (NGS) panel comprising 108 STRs (20 CODIS STRs and 88 non-CODIS STRs), and evaluated its performance in inferring bio-geographical ancestry origins, deconvoluting complex DNA mixtures, and differentiating distant kinships using real and simulated datasets. Our findings demonstrated that incorporating supplementary non-CODIS STRs enabled the extrapolation of multidimensional information from a single STR profile, thereby facilitating the analysis of challenging forensic tasks. In conclusion, this study presents an extensive genomic landscape of forensic non-CODIS STRs among global populations, and emphasized the imperative inclusion of additional polymorphic non-CODIS STRs in future NGS-based forensic systems.

Using two self-developed InDel panels to explore forensic traits and ancestral components in the Hui group

To address the challenges faced by forensic examiners in degraded DNA analysis, we have developed two different panels for various forensic applications, encompassing an AIM-InDel panel for ancestry inference and a Multi-InDel panel for individual identification, respectively. Herein, the efficiencies of these two panels were tested in the Chinese Hui group. By calculating forensic parameters and simulating family relationships, we verified that the Multi-InDel panel could be an effective tool for individual identification, paternity testing, and could assist in kinship identification in the Hui group. For full siblings, the true positive rate of kinship discrimination was 96.553%, when the threshold of log10LR was 1. The cumulative probability of matching as well as cumulative probability of exclusion were 3.8117 × 10−26 and 0.999999722, respectively. Meanwhile, we found that the AIM-InDel panel was effective for bio-geographic ancestry inference, with the vast majority of loci contributing significantly to distinguish East Asian, African, and European populations. By studying the population structure of the Hui ethnic minority, the genetic distance to the Beijing Han population was the closest among the 26 reference populations, which was similar to previous reports. In summary, we have developed two panels which can be effectively applied to the Hui group for individual identification, parentage testing and bio-geographic ancestry inference.

Massively parallel sequencing of 74 microhaplotypes and forensic characteristics in three Chinese Sino-Tibetan populations

Microhaplotype (MH), as an emerging type of forensic genetic marker in recent years, has the potential to support multiple forensic applications, especially for mixture deconvolution and biogeographic ancestry inference. Herein, we investigated the genotype data of 74 MHs included in a novel MH panel, the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel, in three Chinese Sino-Tibetan populations (Han, Tibetan, and Yi) using the Ion Torrent semiconductor sequencing. The sequencing performance, allele frequencies, effective number of alleles (Ae), informativeness (In), and forensic parameters were subsequently estimated and calculated. In addition, principal component analysis (PCA) and structure analysis were performed to explore the population relationships among the three populations and the ancestry component distribution. Overall, this novel MH panel is robust and reliable, and has an excellent sequencing performance. The Ae values ranged from 1.0126 to 7.0855 across all samples, and 75.68 % of MHs had Ae values >2.0000. Allele frequencies at some loci varied considerably among the three studied populations, and the mean In value was 0.0195. Moreover, the genetic affinity between Tibetans and Yis was closer than that between Tibetans and Hans. The aforementioned results suggest that the Ion AmpliSeq MH-74 Plex Microhaplotype Research Panel is highly polymorphic in three investigated populations and could be used as an effective tool for human forensics. Although these 74 MHs have demonstrated the competency in continental population stratification, a higher resolution for distinguishing intracontinental subpopulations and a more comprehensive database with sufficient reference population data still remain to be accomplished.

Comparative evaluation of the MAPlex, Precision ID Ancestry Panel, and VISAGE Basic Tool for biogeographical ancestry inference

Biogeographical ancestry (BGA) inference from ancestry-informative markers (AIMs) has strong potential to support forensic investigations. Over the past two decades, several forensic panels composed of AIMs have been developed to predict ancestry at a continental scale. These panels typically comprise fewer than 200 AIMs and have been designed and tested with a limited set of populations. How well these panels recover patterns of genetic diversity relative to larger sets of markers, and how accurately they infer ancestry of individuals and populations not included in their design remains poorly understood. The lack of comparative studies addressing these aspects makes the selection of appropriate panels for forensic laboratories difficult. In this study, the model-based genetic clustering tool STRUCTURE was used to compare three popular forensic BGA panels: MAPlex, Precision ID Ancestry Panel (PIDAP), and VISAGE Basic Tool (VISAGE BT) relative to a genome-wide reference set of 10k SNPs. The genotypes for all these markers were obtained for a comprehensive set of 3957 individuals from 228 worldwide human populations. Our results indicate that at the broad continental scale (K=6) typically examined in forensic studies, all forensic panels produced similar genetic structure patterns compared to the reference set (G'≈90%) and had high classification performance across all regions (average AUC-PR > 97%). However, at K=7 and K=8, the forensic panels displayed some region-specific clustering deviations from the reference set, particularly in Europe and the region of East and South-East Asia, which may be attributed to differences in the design of the respective panels. Overall, the panel with the most consistent performance in all regions was VISAGE BT with an average weighted AUC̅W score of 96.26% across the three scales of geographical resolution investigated.

Ancestry resolution of South Brazilians by forensic 165 ancestry-informative SNPs panel.

Forensic DNA phenotyping (FDP) includes biogeographic ancestry (BGA) inference and externally visible characteristics (EVCs) prediction directly from an evidential DNA sample as alternatives to provide valuable intelligence when conventional DNA profiling fails to achieve identification. In this context, the application of Massively Parallel Sequencing (MPS) methodologies, which enables simultaneous typing of multiple samples and hundreds of forensic markers, has been gradually implemented in forensic genetic casework. The Precision ID Ancestry Panel (Thermo Fisher Scientific, Waltham, USA) is a forensic multiplex assay consisting of 165 autosomal SNPs designed to provide biogeographic ancestry information. In this work, a sample of 250 individuals from Rio Grande do Sul (RS) State, southern Brazil, apportioned into four main population groups (African-, European-, Amerindian-, and Admixed-derived Gauchos), was evaluated with this panel, to assess the feasibility of this approach in a highly heterogeneous population. Forensic descriptive parameters estimated for each population group revealed that this panel has enough polymorphic and informative SNPs to be used as a supplementary instrument in forensic individual identification and kinship testing regardless of ethnicity. No statistically significant deviation from Hardy-Weinberg equilibrium was observed after Bonferroni correction. However, seven loci pairs displayed linkage disequilibrium in pairwise LD testing (p<3.70× 10-6). Interpopulation comparisons by FST analysis, MDS plot, and STRUCTURE analysis among the four RS population groups apart and along with 89 reference worldwide populations demonstrated that Admixed- and African-derived Gauchos present the highest levels of admixture and population stratification, whereas European- and Amerindian-derived exhibit a more homogeneous genetic conformation.

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 200bp 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.

Recent advances in Forensic DNA Phenotyping of appearance, ancestry and age

Forensic DNA Phenotyping (FDP) comprises the prediction of a person’s externally visible characteristics regarding appearance, biogeographic ancestry and age from DNA of crime scene samples, to provide investigative leads to help find unknown perpetrators that cannot be identified with forensic STR-profiling. In recent years, FDP has advanced considerably in all of its three components, which we summarize in this review article. Appearance prediction from DNA has broadened beyond eye, hair and skin color to additionally comprise other traits such as eyebrow color, freckles, hair structure, hair loss in men, and tall stature. Biogeographic ancestry inference from DNA has progressed from continental ancestry to sub-continental ancestry detection and the resolving of co-ancestry patterns in genetically admixed individuals. Age estimation from DNA has widened beyond blood to more somatic tissues such as saliva and bones as well as new markers and tools for semen. Technological progress has allowed forensically suitable DNA technology with largely increased multiplex capacity for the simultaneous analysis of hundreds of DNA predictors with targeted massively parallel sequencing (MPS). Forensically validated MPS-based FDP tools for predicting from crime scene DNA i) several appearance traits, ii) multi-regional ancestry, iii) several appearance traits together with multi-regional ancestry, and iv) age from different tissue types, are already available. Despite recent advances that will likely increase the impact of FDP in criminal casework in the near future, moving reliable appearance, ancestry and age prediction from crime scene DNA to the level of detail and accuracy police investigators may desire, requires further intensified scientific research together with technical developments and forensic validations as well as the necessary funding.

Multiple founding paternal lineages inferred from the newly-developed 639-plex Y-SNP panel suggested the complex admixture and migration history of Chinese people

BackgroundNon-recombining regions of the Y-chromosome recorded the evolutionary traces of male human populations and are inherited haplotype-dependently and male-specifically. Recent whole Y-chromosome sequencing studies have identified previously unrecognized population divergence, expansion and admixture processes, which promotes a better understanding and application of the observed patterns of Y-chromosome genetic diversity.ResultsHere, we developed one highest-resolution Y-chromosome single nucleotide polymorphism (Y-SNP) panel targeted for uniparental genealogy reconstruction and paternal biogeographical ancestry inference, which included 639 phylogenetically informative SNPs. We genotyped these loci in 1033 Chinese male individuals from 33 ethnolinguistically diverse populations and identified 256 terminal Y-chromosomal lineages with frequency ranging from 0.0010 (singleton) to 0.0687. We identified six dominant common founding lineages associated with different ethnolinguistic backgrounds, which included O2a2b1a1a1a1a1a1a1-M6539, O2a1b1a1a1a1a1a1-F17, O2a2b1a1a1a1a1b1a1b-MF15397, O2a2b2a1b1-A16609, O1b1a1a1a1b2a1a1-F2517, and O2a2b1a1a1a1a1a1-F155. The AMOVA and nucleotide diversity estimates revealed considerable differences and high genetic diversity among ethnolinguistically different populations. We constructed one representative phylogenetic tree among 33 studied populations based on the haplogroup frequency spectrum and sequence variations. Clustering patterns in principal component analysis and multidimensional scaling results showed a genetic differentiation between Tai-Kadai-speaking Li, Mongolic-speaking Mongolian, and other Sinitic-speaking Han Chinese populations. Phylogenetic topology inferred from the BEAST and Network relationships reconstructed from the popART further showed the founding lineages from culturally/linguistically diverse populations, such as C2a/C2b was dominant in Mongolian people and O1a/O1b was dominant in island Li people. We also identified many lineages shared by more than two ethnolinguistically different populations with a high proportion, suggesting their extensive admixture and migration history.ConclusionsOur findings indicated that our developed high-resolution Y-SNP panel included major dominant Y-lineages of Chinese populations from different ethnic groups and geographical regions, which can be used as the primary and powerful tool for forensic practice. We should emphasize the necessity and importance of whole sequencing of more ethnolinguistically different populations, which can help identify more unrecognized population-specific variations for the promotion of Y-chromosome-based forensic applications.

Inferring bio-geographical ancestry with 35 microhaplotypes

During the past decades, with the blooming of large-scale human genetic studies, we are beginning to understand how bio-geographical information could be reflected by genetic variations. And the technological advance in massively parallel sequencing gives advantages to some novel forensic markers such as microhaplotype (BIM). In the present study, we selected and characterized 35 novel bio-geographical informative BIMs based on the 1000 Genomes Project (1KG). All loci had short lengths less than 100 bp, high effective allele numbers (Ae) from 1.875 to 3.980 with an average of 2.798, and high informativeness (In) value from 0.701 to 0.865 with an average of 0.748, which indicates the 35 BIMs possessed great discriminating power. Using these 35 BIMs, the individuals from 1KG could be successfully differentiated into five supergroups defined by 1KG (AFR, AMR, EAS, EUR, and SAS). We also included some individuals from the Simons Genome Diversity Project (SGDP) for further validation. As a result, most individuals could be accurately predicted except for those from super-populations that do not exist in 1KG dataset. In conclusion, the present novel 35 BIMs could be a useful tool for bio-geographical ancestry inference.

Development and validation of a custom panel including 256 Y-SNPs for Chinese Y-chromosomal haplogroups dissection

Y-chromosomal haplogroups determined by Y-chromosomal single nucleotide polymorphisms (Y-SNPs) allow paternal lineage identification and paternal biogeographic ancestry inference, which has attracted a lot of interest in the forensic community. Recently, a comprehensive Y-SNP tool with dominant markers targeting haplogroups in R, E and I branches has been reported, which allows the inference of 640 Y haplogroups. It had a very good performance and could provide a high level of Y haplogroup resolution in most populations. However, the predominant haplogroups in the Chinese populations are O, C and N, suggesting that more Y-SNPs under these clades are needed to achieve the population-specific high resolution. Herein, aiming at the Chinese population, we presented a largely improved custom Y-SNP MPS panel that contains 256 carefully ascertained Y-SNPs based on our previous studies, and evaluated this panel via a series of tests, including the tests for concordance, repeatability, sensitivity, specificity, and stability, as well as the mixture, degraded and case-type sample analysis. The preliminary developmental validation demonstrated that this panel was highly reliable, sensitive, specific, and robust. In the sensitivity test, even when the DNA input was reduced to as low as 0.5 ng, the sample could still be assigned to the correct Y haplogroup. For mixture analysis, even the 1:99 (Male: Female) mixtures had no effects on the assignation of the Y haplogroup of the male contributor. In summary, this assay has provided a high-resolution Y-chromosomal haplogrouping workflow to determine a male’s paternal lineage and/or paternal biogeographic ancestry and could be widely used for Chinese Y-chromosomal haplogroups dissection.

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.

Multivariate statistical approach and machine learning for the evaluation of biogeographical ancestry inference in the forensic field

The biogeographical ancestry (BGA) of a trace or a person/skeleton refers to the component of ethnicity, constituted of biological and cultural elements, that is biologically determined. Nowadays, many individuals are interested in exploring their genealogy, and the capability to distinguish biogeographic information about population groups and subgroups via DNA analysis plays an essential role in several fields such as in forensics. In fact, for investigative and intelligence purposes, it is beneficial to inference the biogeographical origins of perpetrators of crimes or victims of unsolved cold cases when no reference profile from perpetrators or database hits for comparative purposes are available. Current approaches for biogeographical ancestry estimation using SNPs data are usually based on PCA and Structure software. The present study provides an alternative method that involves multivariate data analysis and machine learning strategies to evaluate BGA discriminating power of unknown samples using different commercial panels. Starting from 1000 Genomes project, Simons Genome Diversity Project and Human Genome Diversity Project datasets involving African, American, Asian, European and Oceania individuals, and moving towards further and more geographically restricted populations, powerful multivariate techniques such as Partial Least Squares-Discriminant Analysis (PLS-DA) and machine learning techniques such as XGBoost were employed, and their discriminating power was compared. PLS-DA method provided more robust classifications than XGBoost method, showing that the adopted approach might be an interesting tool for forensic experts to infer BGA information from the DNA profile of unknown individuals, but also highlighting that the commercial forensic panels could be inadequate to discriminate populations at intra-continental level.

Screening and selection of 21 novel microhaplotype markers for ancestry inference in ten Chinese subpopulations

Genetic findings suggested ethnolinguistically diverse populations in China harbor differentiated genetic structure and complex evolutionary admixture histories, providing the genetic basis and theoretical foundation for forensic biogeographical ancestry inference (BGAI). Forensic assays for BGAI among intracontinental eastern Eurasians were previously conducted mainly based on SNPs or InDels. Microhaplotypes, as a set of closely linked SNPs within 200 base pairs, possess the advantages of both STRs and SNPs and have great potential in forensic ancestry inference. However, the forensic assay developed based on ancestry-informative microhaplotypes in the BGAI remains to be further explored, especially in China, which has rich genetic diversity. We described a new BGAI panel based on 21 novel identified ancestry-informative microhaplotypes that focused on dissected finer-scale ancestry compositions of Chinese populations. We initially screened all possible microhaplotypes with high Fst values among five East Asian populations and finally employed 21 candidate microhaplotypes in two multiplex SNaPshot assays. Forensic amplification efficiency and statistically/physically phased haplotypes of the 21 microhaplotypes were validated using SNaPshot and massively parallel sequencing (MPS) platforms. Next, we validated the efficiency of these microhaplotypes for BGAI in 764 individuals from ten Chinese populations using SNaPshot technology. The fine-scale ancestry source and ancestry proportion estimated by principal component analysis (PCA), multidimensional scaling (MDS), phylogenetic tree and model-based STRUCTURE among worldwide populations and East Asians showed that our customized panel could provide a higher discrimination resolution in both continental population stratification and East Asian regional substructure. East Asian populations could be classified into linguistically/geographically different intracontinental subpopulations (Tibeto-Burman, Tai-Kadai and others). Finally, we obtained a higher estimated accuracy using training and tested datasets in the microhaplotype-based panel than traditional SNP-based panels. Generally, the above results demonstrated that this microhaplotype panel was robust and suitable for forensic BGAI in Chinese populations, which provided high discriminatory power for continental populations and discriminated East Asians into linguistically restricted subpopulations.

Genetic insights into the paternal admixture history of Chinese Mongolians via high-resolution customized Y-SNP SNaPshot panels

The Mongolian people, one of the Mongolic-speaking populations, are native to the Mongolian Plateau in North China and southern Siberia. Many ancient DNA studies recently reported extensive population transformations during the Paleolithic to historic periods in this region, while little is known about the paternal genetic legacy of modern geographically different Mongolians. Here, we genotyped 215 Y-chromosomal single nucleotide polymorphisms (Y-SNPs) and 37 Y-chromosomal short tandem repeats (Y-STRs) among 679 Mongolian individuals from Hohhot, Hulunbuir, and Ordos in North China using the AGCU Y37 kit and our developed eight Y-SNP SNaPshot panels (including two panels first reported herein). The C-M130 Y-SNP SNaPshot panel defines 28 subhaplogroups, and the N/O/Q complementary Y-SNP SNaPshot panel defines 30 subhaplogroups of N1b-F2930, N1a1a1a1a3-B197, Q-M242, and O2a2b1a1a1a4a-CTS4658, which improved the resolution our developed Y-SNP SNaPshot panel set and could be applied for dissecting the finer-scale paternal lineages of Mongolic speakers. We found a strong association between Mongolian-prevailing haplogroups and some observed microvariants among the newly generated Y-STR haplotype data, suggesting the possibility of haplogroup prediction based on the distribution of Y-STR haplotypes. We identified three main ancestral sources of the observed Mongolian-dominant haplogroups, including the local lineage of C2*-M217 and incoming lineages from other regions of southern East Asia (O2*-M122, O1b*-P31, and N1*-CTS3750) and western Eurasia (R1*-M173). We also observed DE-M145, D1*-M174, C1*-F3393, G*-M201, I-M170, J*-M304, L-M20, O1a*-M119, and Q*-M242 at relatively low frequencies (< 5.00%), suggesting a complex admixture history between Mongolians and other incoming Eurasians from surrounding regions. Genetic clustering analyses indicated that the studied Mongolians showed close genetic affinities with other Altaic-speaking populations and Sinitic-speaking Hui people. The Y-SNP haplotype/haplogroup-based genetic legacy not only revealed that the stratification among geographically/linguistically/ethnically different Chinese populations was highly consistent with the geographical division and language classification, but also demonstrated that patrilineal genetic materials could provide fine-scale genetic structures among geographically different Mongolian people, suggesting that our developed high-resolution Y-SNP SNaPshot panels have the potential for forensic pedigree searches and biogeographical ancestry inference.

Massively parallel sequencing of 165 ancestry-informative SNPs and forensic biogeographical ancestry inference in three southern Chinese Sinitic/Tai-Kadai populations

Ancestry informative markers (AIMs), which are distributed throughout the human genome, harbor significant allele frequency differences among diverse ethnic groups. The use of sets of AIMs to reconstruct population history and genetic relationships is attracting interest in the forensic community, because biogeographic ancestry information for a casework sample can potentially be predicted and used to guide the investigative process. However, subpopulation ancestry inference within East Asia remains in its infancy due to a lack of population reference data collection and incomplete validation work on newly developed or commercial AIM sets. In the present study, 316 Chinese persons, including 85 Sinitic-speaking Haikou Han, 120 Qiongzhong Hlai and 111 Daozhen Gelao individuals belonging to Tai-Kadai-speaking populations, were analyzed using the Precision ID Ancestry Panel (165 AISNPs). Combined with our previous 165-AISNP data (375 individuals from 6 populations), the 1000 Genomes Project and forensic literature, comprehensive population genetic comparisons and ancestry inference were further performed via ADMIXTURE, TreeMix, PCA, f-statistics and N-J tree. Although several nonpolymorphic loci were identified in the three southern Chinese populations, the forensic parameters of this ancestry inference panel were better than those for the 23 STR-based Huaxia Platinum System, which is suitable for use as a robust tool in forensic individual identification and parentage testing. The results based on the ancestry assignment and admixture proportion evaluation revealed that this panel could be used successfully to assign individuals at a continental scale but also possessed obvious limitations in discriminatory power in intercontinental individuals, especially for European-Asian admixed Uyghurs or in populations lacking reference databases. Population genetic analyses further revealed five continental population clusters and three East Asian-focused population subgroups, which is consistent with linguistic affiliations. Ancestry composition and multiple phylogenetic analysis further demonstrated that the geographically isolated Qiongzhong Hlai harbored a close phylogenetic relationship with Austronesian speakers and possessed a homogenous Tai-Kadai-dominant ancestry, which could be used as the ancestral source proxy in population history reconstruction of Tai-Kadai-speaking populations and as one of the representatives for forensic database establishment. In summary, more population-specific AIM sets focused on East Asian subpopulations, comprehensive algorithms and high-coverage population reference data should be developed and validated in the next step.