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.     

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.     

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.     

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.     

EurEAs_Gplex—A new SNaPshot assay for continental population discrimination and gender identification

Assays that allow analysis of the biogeographic origin of biological samples in a standard forensic laboratory have to target a small number of highly differentiating markers. Such markers should be easy to multiplex and the assay must perform well in the degraded and scarce biological material. SNPs localized in the genome regions, which in the past were subjected to differential selective pressure in various populations, are the most widely used markers in the studies of biogeographic affiliation. SNPs reflecting biogeographic differences not related to any phenotypic traits are not sufficiently explored.The goal of our study was to identify a small set of SNPs not related to any known pigmentation/phenotype-specific genes, which would allow efficient discrimination between populations of Europe and East Asia. The selection of SNPs was based on the comparative analysis of representative European and Chinese/Japanese samples (B-lymphocyte cell lines), genotyped using the Infinium HumanOmniExpressExome microarray (Illumina). The classifier, consisting of 24 unlinked SNPs (24-SNP classifier), was selected. The performance of a 14-SNP subset of this classifier (14-SNP subclassifier) was tested using genotype data from several populations. The 14-SNP subclassifier differentiated East Asians, Europeans and Africans with ∼100% accuracy; Palestinians, representative of the Middle East, clustered with Europeans, while Amerindians and Pakistani were placed between East Asian and European populations.Based on these results, we have developed a SNaPshot assay (EurEAs_Gplex) for genotyping SNPs from the 14-SNP subclassifier, combined with an additional marker for gender identification. Forensic utility of the EurEAs_Gplex was verified using degraded and low quantity DNA samples. The performance of the EurEAs_Gplex was satisfactory when using degraded DNA; tests using low quantity DNA samples revealed a previously not described source of genotyping errors, potentially important for any SNaPshot-based assays.     

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.     

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.     

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.     

Prediction of biogeographical ancestry from genotype: a comparison of classifiers

DNA can provide forensic intelligence regarding a donor’s biogeographical ancestry (BGA) and other externally visible characteristics (EVCs). A number of algorithms have been proposed to assign individual human genotypes to a BGA using ancestry informative marker (AIM) panels. This study compares the BGA assignment accuracy of the population clustering program STRUCTURE and three generic classification approaches including a Bayesian algorithm, genetic distance, and multinomial logistic regression (MLR). A selection of 142 ancestry informative single nucleotide polymorphisms (SNPs) were chosen from existing marker panels (SNPforID 34-plex, Eurasiaplex, Seldin, and Kidd’s AIM panels) to assess BGA classification at the continental level for Africans, Europeans, East Asians, and Amerindians. A training set of 1093 individuals with self-declared BGA from the 1000 Genomes phase 1 database was used by each classifier to predict BGA in a test set of 516 individuals from the HGDP-CEPH (Stanford) cell line panel. Tests were repeated with 0, 10, 50, 70, and 90% of the genotypes missing. Comparison of the area under the receiver operating characteristic curves (AUROCs) showed high accuracy in STRUCTURE and the generic Bayesian approach. The latter algorithm offers a computationally simpler alternative to STRUCTURE with little loss in accuracy and is suitable for phenotype prediction while STRUCTURE is not.

     

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

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

Development and 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.     

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.     

A comprehensive exploration of the genetic legacy and forensic features of Afghanistan and Pakistan Mongolian-descent Hazara

Afghanistan and Pakistan are rich with a complex landscape of culture, linguistics, ethnicity and genetic legacy at the crossroads between Indian-Subcontinent and Central Asia. Hazara people have historically been suggested to be Mongolian decedents but seldom been genetically studied. To dissect the genetic structure and explore the forensic characteristics of Hazara people, we first genotyped 30 Insertion/deletion (Indel) markers in 468 samples from 2 aboriginal Hazara populations from Afghanistan and Pakistan, and 100 East Asian comparative Bouyei samples using the Investigator® DIPplex kit. Subsequently, we carried out a comprehensive population genetic analysis from four different datasets: 8895 30-Indel genotype data from 51 populations, 15,895 30-Indel allele frequency data from 98 populations, 1048 genotypes of 993 STRs and Indels from 53 HGDP populations and 2068 whole-genomes (621,799 single nucleotide polymorphisms) from 165 worldwide Human origin reference populations, to further unravel the genetic complexity between Hazara and worldwide human populations using various statistical analysis. We find that 30 Indels are in accordance with HWE, and informative and polymorphic in both Central Asians Hazara and East Asian Bouyei populations. The forensic combined probability of exclusion is larger than 0.9943 and the cumulative power of discrimination is larger than 0.99999999999936. These forensic parameters show the high level of diversity, which makes the Indel amplification system suitable for forensic routine work and may be used as a supplementary assay for routine forensic investigation. The results from pairwise genetic distances, MDS, PCA, and phylogenetic relationship reconstruction demonstrate that present-day Hazaras are genetically closer to the Turkic-speaking populations (Uyghur, Kazakh, and Kyrgyz) residing in northwest China than with other Central/South Asian populations and Mongolian. Outgroup and admixture f_3, f₄, f₄-ratio, qpWave, and qpAdm results further demonstrate that Hazara shares more alleles with East Asians than with other Central Asians and carries 57.8% Mongolian-related ancestry. Overall, our findings suggest that Hazaras have experienced genetic admixture with the local or neighboring populations and formed the current East-West Eurasian admixed genetic profile after their separation from the Mongolians.     

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.     

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.     

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

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

EASplex: A panel of 308 AISNPs for East Asian ancestry inference using next generation sequencing

Ancestry inference is useful in many scientific fields, such as forensic genetics, medical genetics, and molecular archaeology. Various ancestry inferring methods have been released for major continental populations. However, few reports refer to sub-populations within the East Asian population using hundreds of ancestry informative SNPs (AISNPs). In this study, we developed a 308-AISNP panel (EASplex NGS DNA panel) using multiplex PCR and next generation sequencing (NGS). This panel included 56 SNPs relevant for the continent-level ancestry inference and 252 Japanese-, Korean-, and/or Han Chinese-specific AISNPs to address the ancestry inference of global populations and regional populations among Japanese (JPT), Korean minority (CHK), and Han Chinese (CHH). A total of 87 CHK and 59 CHH samples were used to check the performance of the EASplex NGS DNA panel. By analyzing 146 profiles of samples with JPT and CHH data from Beijing and South China in 1000 genomes project, the following results were obtained: (1) the 146 tested samples were correctly assigned to the East Asian group; (2) the paired population assignment rate was 99.73% for JPT and CHH, 95% for JPT and CHK, and 90.11% for CHK and CHH; and (3) the whole population assignment was 92.14% for the JPT, CHK, and CHH data. Overall, the EASplex NGS DNA panel displayed informativeness for continental ancestry inference and regional ancestry inference among JPT, CHH, and CHK and has the potential for use in forensic and genetic population studies.     

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

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

A Japanese-specific allele in the GALNT11 gene

In this study, five single nucleotide polymorphisms (SNPs) in the ABCC4, FBN1, CEP152, ZNF804B, and GALNT11 genes were investigated to assess allele frequencies in 14 different populations by a novel pentaplex PCR method. All SNPs were polymorphic in East Asians, whereas mutant alleles were absent or rare in non-East Asians. The frequencies of a mutant allele in FBN1 (rs140598) showed a north–south downward cline in East Asia, whereas those of a mutant allele in ZNF804B (rs1916830) were relatively uniform in East Asia. The highest frequencies of mutant alleles in ABCC4 (rs3765534), CEP152 (rs2289178), and GALNT11 (rs3778922) were observed in Okinawa. The mutant allele in GALNT11 was found only in Far-East Asian populations: the frequencies were about 0.153 in Okinawa, 0.076 in the main island of Japan, and 0.017–0.004 in Korea. These five East Asian- and Japanese-specific SNPs would be useful markers for forensic individualization, in particular, as ancestry-informative markers.     

Application of six IrisPlex SNPs and comparison of two eye color prediction systems in diverse Eurasia populations

DNA-based prediction for externally visible characteristics such as eye color is already a useful tool in forensic criminal investigations. The IrisPlex system, consisting of six single nucleotide polymorphisms (SNPs) and a prediction model, was developed based on individuals from several European populations. Other recent studies have developed a different prediction model, also based on European populations. In this study, we compared two prediction models using the data for the six IrisPlex SNPs genotyped on 905 individuals from 12 different Eurasian populations. All SNPs showed significant differences in allele frequencies among three groups of populations: European, genetically intermediate (Khanty, Uygur, and Yakut), and East Asian. The two prediction models, the FROG-kb calculation based on the formula of Walsh et al. (2011) and the Snipper calculation from Ruiz et al. (2013), gave identical predictions of brown eye color for the four East Asian populations with complete data but did not give concordant predictions for many individuals in the seven intermediate and European populations. Inconsistencies were mainly conclusive prediction by one model but not the other. Of the 714 individuals with complete 6-locus genotypes, the two models gave 22 % inconsistent predictions. Eliminating the 306 individuals in the Korean and three Chinese populations, in which the predictions were always consistent for brown eye color, the inconsistencies (among the remaining 408 individuals) were 38.7 %. We conclude that more attention should be paid to predictive uncertainty/error. Implementation of both prediction models in future forensic casework is one immediate way to highlight uncertainty.