Metabolic diversity in human populations and correlation with genetic and ancestral geographic distances.

DNA polymorphic markers and self-defined ethnicity groupings are used to group individuals with shared ancient geographic ancestry. Here we studied whether ancestral relationships between individuals could be identified from metabolic screening data reported by the California newborn screening (NBS) program. NBS data includes 41 blood metabolites measured by tandem mass spectrometry from singleton babies in 17 parent-reported ethnicity groupings. Ethnicity-associated differences identified for 71% of NBS metabolites (29 of 41, Cohen's d > 0.5) showed larger differences in blood levels of acylcarnitines than of amino acids (P < 1e-4). A metabolic distance measure, developed to compare ethnic groupings based on metabolic differences, showed low positive correlation with genetic and ancient geographic distances between the groups' ancestral world populations. Several outlier group pairs were identified with larger genetic and smaller metabolic distances (Black versus White) or with smaller genetic and larger metabolic distances (Chinese versus Japanese) indicating the influence of genetic and of environmental factors on metabolism. Using machine learning, comparison of metabolic profiles between all pairs of ethnic groupings distinguished individuals with larger genetic distance (Black versus Chinese, AUC = 0.96), while genetically more similar individuals could not be separated metabolically (Hispanic versus Native American, AUC = 0.51). Additionally, we identified metabolites informative for inferring metabolic ancestry in individuals from genetically similar populations, which included biomarkers for inborn metabolic disorders (C10:1, C12:1, C3, C5OH, Leucine-Isoleucine). This work sheds new light on metabolic differences in healthy newborns in diverse populations, which could have implications for improving genetic disease screening.

State of the Art for Microhaplotypes.

In recent years, the number of publications on microhaplotypes has averaged more than a dozen papers annually. Many have contributed to a significant increase in the number of highly polymorphic microhaplotype loci. This increase allows microhaplotypes to be very informative in four main areas of forensic uses of DNA: individualization, ancestry inference, kinship analysis, and mixture deconvolution. The random match Probability (RMP) can be as small as 10−100 for a large panel of microhaplotypes. It is possible to measure the heterozygosity of an MH as the effective number of alleles (Ae). Ae > 7.5 exists for African populations and >4.5 exists for Native American populations for a smaller panel of two dozen selected microhaplotypes. Using STRUCTURE, at least 10 different ancestral clusters can be defined by microhaplotypes. The Ae for a locus is also identical to the Paternity Index (PI), the measure of how informative a locus will be in parentage testing. High Ae loci can also be useful in missing persons cases. Finally, high Ae microhaplotypes allow the near certainty of seeing multiple additional alleles in a mixture of two or more individuals in a DNA sample. In summary, a panel of higher Ae microhaplotypes can outperform the standard CODIS markers.

A multipurpose panel of microhaplotypes for use with STR markers in casework.

A small panel of highly informative loci that can be genotyped on the same equipment as the standard CODIS short tandem repeat (STR) markers has strong potential for application in forensic casework. Single nucleotide polymorphisms (SNPs) can be typed by a couple of methods on capillary electrophoresis (CE) machines and on sequencers, but the amount of information relative to the laboratory effort has hindered use of SNPs in actual casework. Insertion-deletion markers (InDels) suffer from similar problems. Microhaplotypes (MHs) are much more informative per locus but have similar technical difficulties unless they are typed by massively parallel sequencing (MPS). As forensic labs are acquiring sequencing machines, MHs become more likely to be used in casework, especially if multiplexed with STRs. Here we present the details of a multipurpose panel of 24 MHs with the highest effective number of alleles (Ae) from previous work. An augmented STR panel of 24 loci (20 CODIS markers plus four commonly typed STRs) is also considered. The Ae and ancestry informativeness (In) distributions of these two datasets are compared. The MH panel is shown to have better individualization and population distinction than the augmented CODIS STRs. We note that the 24 MHs should be better for mixture analyses than the STRs. Finally, we suggest that a commercial kit including both the standard CODIS markers and this set of 24 MH would greatly improve the discrimination power over that of current commercial assays.

North Asian population relationships in a global context.

Population genetic studies of North Asian ethnic groups have focused on genetic variation of sex chromosomes and mitochondria. Studies of the extensive variation available from autosomal variation have appeared infrequently. We focus on relationships among population samples using new North Asia microhaplotype data. We combined genotypes from our laboratory on 58 microhaplotypes, distributed across 18 autosomes, on 3945 individuals from 75 populations with corresponding data extracted for 26 populations from the Thousand Genomes consortium and for 22 populations from the GenomeAsia 100 K project. A total of 7107 individuals in 122 total populations are analyzed using STRUCTURE, Principal Component Analysis, and phylogenetic tree analyses. North Asia populations sampled in Mongolia include: Buryats, Mongolians, Altai Kazakhs, and Tsaatans. Available Siberians include samples of Yakut, Khanty, and Komi Zyriane. Analyses of all 122 populations confirm many known relationships and show that most populations from North Asia form a cluster distinct from all other groups. Refinement of analyses on smaller subsets of populations reinforces the distinctiveness of North Asia and shows that the North Asia cluster identifies a region that is ancestral to Native Americans.

New Insight into the human genetic diversity in North African populations by genotyping of SNPs in DRD3, CSMD1 and NRG1 genes.

BACKGROUND: The single nucleotide polymorphisms (SNPs) of the dopamine D3 receptor (DRD3), the CUB and sushi multiple domains 1 (CSMD1) and the neuregulin 1 (NRG1) genes were used to study the genetic diversity and affinity among North African populations and to examine their genetic relationships in worldwide populations. METHODS: The rs3773678, rs3732783 and rs6280 SNPs of the DRD3 gene located on chromosome 3, the rs10108270 SNP of the CSMD1 gene and the rs383632, rs385396 and rs1462906 SNPs of the NRG1 gene located on chromosome 8 were analysed in 366 individuals from seven North African populations (Libya, Kairouan, Mehdia, Sousse, Kesra, Smar and Kerkennah). RESULTS: The low values of FST indicated that only 0.27%-1.65% of the genetic variability was due to the differences between the populations. The Kairouan population has the lowest average heterozygosity among the North African populations. Haplotypes composed of the ancestral alleles ACC and ACAT were more frequent in the Kairouan population than in other North African populations. The PCA and the haplotypic analysis showed that the genetic structure of populations in North Africa was closer to that of Europeans, Admixed Americans, South Asians and East Asians. However, analysis of the rs3732783 and rs6280 SNPs revealed that the CT microhaplotype was specific to the North African population. CONCLUSIONS: The Kairouan population exhibited a relatively low rate of genetic variability. The North African population has undergone significant gene flow but also evolutionary forces that have made it genetically distinct from other populations.

The population genetics characteristics of a 90 locus panel of microhaplotypes.

Single-nucleotide polymorphisms (SNPs) and small genomic regions with multiple SNPs (microhaplotypes, MHs) are rapidly emerging as novel forensic investigative tools to assist in individual identification, kinship analyses, ancestry inference, and deconvolution of DNA mixtures. Here, we analyzed information for 90 microhaplotype loci in 4009 individuals from 79 world populations in 6 major biogeographic regions. The study included multiplex microhaplotype sequencing (mMHseq) data analyzed for 524 individuals from 16 populations and genotype data for 3485 individuals from 63 populations curated from public repositories. Analyses of the 79 populations revealed excellent characteristics for this 90-plex MH panel for various forensic applications achieving an overall average effective number of allele values (Ae) of 4.55 (range 1.04-19.27) for individualization and mixture deconvolution. Population-specific random match probabilities ranged from a low of 10-115 to a maximum of 10-66. Mean informativeness (In) for ancestry inference was 0.355 (range 0.117-0.883). 65 novel SNPs were detected in 39 of the MHs using mMHseq. Of the 3018 different microhaplotype alleles identified, 1337 occurred at frequencies > 5% in at least one of the populations studied. The 90-plex MH panel enables effective differentiation of population groupings for major biogeographic regions as well as delineation of distinct subgroupings within regions. Open-source, web-based software is available to support validation of this technology for forensic case work analysis and to tailor MH analysis for specific geographical regions.

STAT3 polymorphisms in North Africa and its implication in breast cancer.

BACKGROUND: Only a few studies have investigated the association of single nucleotide polymorphisms in STAT3 gene with the susceptibility to cancer and response to chemotherapy. Our aim was to determine the allele frequencies of rs3869550, rs957971, and rs7211777 at the STAT3 gene in North African populations and compare them to 1000 genomes populations, and to investigate their relation with cancer. METHODS: The targeted SNPs have been analyzed in six Tunisian populations and a sample of Libyans using TaqMan® Assay. The results were compared to 1000 Genomes Project population samples. Targeting of the regions encompassing the three SNPs by micro-ARN was assessed using miR databases. RESULTS: The analysis of the 3 SNPs showed that North African populations were close to South Asians. As expected, African populations presented a significant frequency of the ancestral CCG haplotype in contrast to other populations where the fully derived TGA haplotype was more frequent. The presence and diversity of rare haplotypes at STAT3 in North African populations could have been generated by recombination between the two major haplotypes. A screening of the micro-RNA databases showed that the STAT3 region with the mutated allele of rs7211777 (G>A) could be targeted by miR hsa-miR-3606-5p, which also targets genes involved in breast cancer.

Genetic relationships of Southwest Asian and Mediterranean populations.

The Southwest Asian, circum-Mediterranean, and Southern European populations (collectively, SWAMSE) together with Northern European populations form one of five "continental" groups of global populations in many analyses of population relationships. This region is of great anthropologic and forensic interest but relationships of large numbers of populations within the region have not been able to be cleanly resolved with autosomal genetic markers. To examine the genetic boundaries to the SWAMSE region and whether internal structure can be detected we have assembled data for a total of 151 separate autosomal genetic markers on populations in this region and other parts of the world for a global set of 95 populations. The markers include 83 ancestry informative SNPs as singletons and 68 microhaplotype loci defined by 204 SNPs. The 151 loci are ancestry informative on a global scale, identifying at least five biogeographic clusters. One of those clusters is a clear grouping of 37 populations containing the SWAMSE plus northern European populations to the exclusion of populations in South Central Asia and populations from farther East. A refined analysis of the 37 populations shows the northern European populations clustering separately from the SWAMSE populations. Within Southwest Asia the Samaritans and Shabaks are distinct outliers. The Yemenite Jews, Saudi, Kuwaiti, Palestinian Arabs, and Southern Tunisians cluster together loosely while the remaining populations from Northern Iraq, Mediterranean Europe, the Caucasus region, and Iran cluster in a more complex graded fashion. The majority of the SWAMSE populations from the mainland of Southwest Asia form a cluster with little internal structure reflecting a very complex history of endogamy and migrations. The set of 151 DNA polymorphisms not only distinguishes major geographical regions globally but can distinguish ancestry to a small degree within geographical regions such as SWAMSE. We discuss forensic characteristics of the polymorphisms and also identify those that rank highest by Rosenberg's In measure for the SWAMSE region populations and for the global set of populations analyzed. DATA AVAILABILITY: Genotypes on all 151 markers for all 3790 individuals typed in the Kidd Lab on the 72 Kidd lab populations have been deposited in the Zenodo archive and can be freely accessed at https://doi.org/10.5281/zenodo.4658892. Some of the data has been made public previously as supplemental files appended to publications. Data for the additional individuals included in the analyses was taken from already public datasets as indicated in the text.

Genetic diversity of the North African population revealed by the typing of SNPs in the DRD2/ANKK1 genomic region.

The dopamine - related genes, like dopamine D2 receptor (DRD2) gene and ankyrin repeat and kinase domain containing 1 (ANKK1) gene are implicated in neurological functions. Some polymorphisms of the DRD2/ANKK1 locus (TaqIA, TaqIB, TaqID) have been used to study genetic diversity and the evolution of human populations. The present investigation aims to assess the genetic diversity in seven North African populations in order to explore their genetic structure and to compare them to others worldwide populations studied for the same locus. Nine single nucleotide polymorphisms (SNPs) from the DRD2/ANKK1 locus (rs1800497 TaqIA, rs2242592, rs1124492, rs6277, rs6275, rs1079727, rs2002453, rs2234690 and rs1079597 TaqIB) were typed in 366 individuals from seven North African populations: six from Tunisia (Sousse, Smar, Kesra, Kairouan, Mehdia and Kerkennah) and one from Libya. The allelic frequencies of rs2002453 and rs2234690 were higher in the Smar population than in the other North African populations. More, the Smar population showed the lowest average heterozygosity (0.313). The principal component analysis (PCA) showed that the Smar population was clearly separated from others. Furthermore, linkage disequilibrium analysis shown a high linkage disequilibrium in the North African population and essentially in Smar population. Comparison with other world populations has shown that the heterozygosity of North African population was very close to that of the African and European populations. The PCA and the haplotypic analysis suggested the presence of an important Eurasian genetic component for the North African population. These results suggested that the Smar population was isolated from the others North Africans ones by its peculiar genetic structure because of isolation, endogamy and genetic drift. On the other hand, the North African population is characterized by a multi ancestral gene pool from Eurasia and sub-Saharan Africa due to human migration since prehistoric times.

A Distinctive Pattern of Diversity for the TAS2R38 Gene in North Africa.

The TAS2R38 gene is involved in bitter taste perception. This study documents the distinctive diversity patterns in northern Africa of functional single-nucleotide polymorphisms (SNPs) rs713598 and rs1726866 at the TAS2R38 locus and places those patterns in the context of global TAS2R38 diversity. Data previously genotyped with TaqMan assay were analyzed for rs713598 and rs1726866 for 375 unrelated subjects (305 Tunisians from seven locations: Mahdia, Sousse, Kesra, Nebeur, Kairouan, Smar, and Kerkennah; plus 70 Libyans). Data were analyzed to present haplotypes and genotypes before comparison with data from worldwide populations. This study provides information about TAS2R38 diversity in a part of the world that is relatively understudied. Considering the two SNPs rs713598 and rs1726866, the CA nucleotide haplotype leading to the PV amino acid haplotype is extremely rare almost everywhere, but it is relatively frequent (between 6% and 15%) in northern Africa, where it coexists with the globally common amino acid haplotypes PA, AA, and AV. Given its higher frequency in North Africa, the authors propose the CA nucleotide haplotype as a biogeographic marker for forensic purposes.

The distinctive geographic patterns of common pigmentation variants at the OCA2 gene.

Oculocutaneous Albinism type 2 (OCA2) is a gene of great interest because of genetic variation affecting normal pigmentation variation in humans. The diverse geographic patterns for variant frequencies at OCA2 have been evident but have not been systematically investigated, especially outside of Europe. Here we examine population genetic variation in and near the OCA2 gene from a worldwide perspective. The very different patterns of genetic variation found across world regions suggest strong selection effects may have been at work over time. For example, analyses involving the variants that affect pigmentation of the iris argue that the derived allele of the rs1800407 single nucleotide polymorphism, which produces a hypomorphic protein, may have contributed to the previously demonstrated positive selection in Europe for the enhancer variant responsible for light eye color. More study is needed on the relationships of the genetic variation at OCA2 to variation in pigmentation in areas beyond Europe.

Validation of novel forensic DNA markers using multiplex microhaplotype sequencing.

Microhaplotypes (MH) are comprised of multiple single nucleotide polymorphisms (SNPs) that are located within 300 bases of genomic sequence. Improved tools are needed to facilitate broader application of microhaplotypes in a diverse range of populations and forensic settings. We designed an assay for multiplex sequencing of 90 microhaplotypes (mMHseq) that include 46 MH loci with high Effective Number of Alleles (Ae) from previous studies [1], and 44 high Ae MH loci containing between four to fourteen SNPs that were identified from the 1000 Genomes (1KG) Project. The unique design of mMHseq integrates a novel method for multiplex amplification from small DNA amounts, and multiplex sequencing of 48 samples in a single MiSeq run to detect all relevant MH variation. Assay performance was evaluated in a cohort of 156 individuals from seven different world populations from Africa, Asia, and Europe. Three of those populations from East Africa (Chagga, Sandawe, and Zaramo) and one from Eastern Europe (Adygei) had sufficient individuals sequenced by the assay to be included in statistical analyses with the 26 1KG populations. For those 30 populations the mean global average Ae was 5.08 (range: 2.7-11.54) and mean informativeness for biogeographic variation (In) was 0.30 (range: 0.08-0.70). Eighty-five novel SNPs were detected in 58 of the 90 microhaplotypes. Open-source, web-based software was developed to visualize haplotype phase data for each microhaplotype and individual. Our approach for multiplex microhaplotype sequencing can be customized and expanded as novel loci are being discovered.

FrogAncestryCalc: A standalone batch likelihood computation tool for ancestry inference panels catalogued in FROG-kb.

The web-based application, FROG-kb (the Forensic Resource/Reference on Genetics-knowledge base, https://frog.med.yale.edu) supports the use of Single Nucleotide Polymorphisms (SNPs) for individual identification and ancestry inference in a forensic setting. The primary functionality provided by FROG-kb on the web is computation of relative likelihoods of populations being the origin of an individual, utilizing the underlying reference population allele frequency data curated and organized in ALFRED, the ALlele FREquency Database (https://alfred.med.yale.edu/). Here we present a downloadable stand-alone tool, FrogAncestryCalc that can simultaneously compute population likelihoods for multiple individuals for a selected panel of SNPs. The program calculates for a given Ancestry Inference (AI) panel the probability of each individual's genotype profile arising in each of the reference populations. Five of the AI panels catalogued in FROG-kb are implemented in this version of FrogAncestryCalc.

Population relationships based on 170 ancestry SNPs from the combined Kidd and Seldin panels.

The benefits of ancestry informative SNP (AISNP) panels can best accrue and be properly evaluated only as sufficient reference population data become readily accessible. Ideally the set of reference populations should approximate the genetic diversity of human populations worldwide. The Kidd and Seldin AISNP sets are two panels that have separately accumulated thus far the largest and most diverse collections of data on human reference populations from the major continental regions. A recent tally in the ALFRED allele frequency database finds 164 reference populations available for all the 55 Kidd AISNPs and 132 reference populations for all the 128 Seldin AISNPs. Although much more of the genetic diversity in human populations around the world still needs to be documented, 81 populations have genotype data available for all 170 AISNPs in the union of the Kidd and Seldin panels. In this report we examine admixture and principal component analyses on these 81 worldwide populations and some regional subsets of these reference populations to determine how well the combined panel illuminates population relationships. Analyses of this dataset that focused on Native American populations revealed very strong cluster patterns associated with many of the individual populations studied.

Genetic relationships of European, Mediterranean, and SW Asian populations using a panel of 55 AISNPs.

The set of 55 ancestry informative SNPs (AISNPs) originally developed by the Kidd Lab has been studied on a large number of populations and continues to be applied to new population samples. The existing reference database of population samples allows the relationships of new population samples to be inferred on a global level. Analyses show that these autosomal markers constitute one of the better panels of AISNPs. Continuing to build this reference database enhances its value. Because more than half of the 25 ethnic groups recently studied with these AISNPs are from Southwest Asia and the Mediterranean region, we present here various analyses focused on populations from these regions along with selected reference populations from nearby regions where genotype data are available. Many of these ethnic groups have not been previously studied for forensic markers. Data on populations from other world regions have also been added to the database but are not included in these focused analyses. The new population samples added to ALFRED and FROG-kb increase the total to 164 population samples that have been studied for all 55 AISNPs.

Usefulness of COMT gene polymorphisms in North African populations.

The COMT gene encodes for catechol-O-methyl-transferase, an enzyme playing a major role in regulation of synaptic catecholamine neurotransmitters. Investigating 4 markers of the COMT gene (rs2020917, rs4818, rs4680, rs9332377) in 6 Tunisian populations and a pool of Libyans. Our objective was to determine the distribution of allelic, genotypic and haplotypic frequencies by comparison to other populations of the 1000 genomes project and 59 populations from the Kidd Lab dataset. The allelic frequencies established for these SNPs in the North African populations are similar to those of Europeans and South Asians. Linkage disequilibrium between these SNPs and haplotypes frequencies are different between populations whose clustering in principal components analysis (PCA) according to their geographic origin was more significant using haplotypic frequencies. COMT activity prediction by haplotypes genotyping could be limited to rs4818-rs4680 micro-haplotypes. The Low activity haplotype (CG) displays the highest frequency in African populations (55%), in the 59 Kidd Lab populations we found also that Sub-Saharan Africans, Native Americans, and some East Asian and Pacific Island populations all have frequencies in the 50-81% range for (CG) where as its lowest frequency was found in Europeans (10%), this results have been also confirmed for Southwest Asians. North Africans and South Asians with intermediate frequencies have approximately similar values (20% and 25%). Europeans show the highest frequencies of haplotypes with predicted High and Medium activity in contrast to Africans. North Africans and South Asians present similar results for all the category of the COMT activity prediction by haplotypes genotyping. The high level of genetic diversity of COMT haplotypes, not only allows distinction between populations according to their history settlement, origin and ethnicity, it constitutes a basis for studies of association of the COMT gene polymorphism with pathologies, drugs response and for forensic investigation in North African populations.

Recent Selection on a Class I ADH Locus Distinguishes Southwest Asian Populations Including Ashkenazi Jews.

The derived human alcohol dehydrogenase (ADH)1B*48His allele of the ADH1B Arg48His polymorphism (rs1229984) has been identified as one component of an East Asian specific core haplotype that underwent recent positive selection. Our study has been extended to Southwest Asia and additional markers in East Asia. Fst values (Sewall Wright's fixation index) and long-range haplotype analyses identify a strong signature of selection not only in East Asian but also in Southwest Asian populations. However, except for the ADH2B*48His allele, different core haplotypes occur in Southwest Asia compared to East Asia and the extended haplotypes also differ. Thus, the ADH1B*48His allele, as part of a core haplotype of 10 kb, has undergone recent rapid increases in frequency independently in the two regions after divergence of the respective populations. Emergence of agriculture may be the common factor underlying the evident selection.

Selecting microhaplotypes optimized for different purposes.

Massively parallel sequencing is transforming forensic work by allowing various useful forensic markers, such as STRPs and SNPs, to be multiplexed providing information on ancestry, individual and familial identification, phenotypes for eye/hair/skin pigmentation, and the deconvolution of mixtures. Microhaplotypes also become feasible with massively parallel sequencing, these are DNA segments (smaller than 300 nucleotides) that are selected to contain multiple SNPs unambiguously defining three or more haplotype alleles occurring at common frequencies. The physical extent of a microhaplotype can thus be covered by a single sequence read making these loci phase-known codominant genetic systems. Such microhaplotypes supply significantly more information than a single SNP can. Our efforts to develop useful sets of microhaplotypes have already identified 182 such loci that we have studied on a large number of human populations from around the world. We present various analyses on 83 populations in our ongoing study for a subset of the best microhaplotypes currently available illustrating their characteristics and potential utility for ancestry, identification, and mixture deconvolution.

Improving ancestry distinctions among Southwest Asian populations.

The Kidd Lab panel of 55 AISNPs can provide up to 10 statistically relevant biogeographic groupings of a global set of populations. A second-tier panel would be useful for increasing the accuracy for further differentiation of populations within a specific global grouping. Because recent advances in massively parallel sequencing (MPS) methods allow the genotyping of many more SNPs, we are now identifying additional SNPs to provide refined discrimination among regional subsets of populations; Southwest Asia and the nearby Mediterranean region (SWA) is our current target for such a "second tier" panel. We selected the potentially best SNPs from various sources: our own laboratory database (>4600 SNPs), AISNP panels (Kidd 55 and Seldin 128 SNP panels), and published papers reporting European and SW Asian populations. Rosenberg's Informativeness, Fst, and allele frequency heatmap matrices are used to determine the best SNPs for the region. A total of 2568 individuals, from 39 different populations ranging from North-East Africa through the SW Asia and Europe to the Ural Mountains, were included in the refinement processes and analyses. Heatmap, PCA, Structure (K = 4), and ancestry inference for selected individuals with an in-lab version of FROG-kb analyses indicate that these 86 AISNPs provide the basis for building an improved, optimized panel of AISNPs that collectively provide additional information on differences among populations in that part of the world. Testing this panel with additional populations from the area and with new SNPs and/or microhaplotypes is expected to improve the panel.

The redesigned Forensic Research/Reference on Genetics-knowledge base, FROG-kb.

The Forensic Resource/Reference on Genetics-knowledge base (FROG-kb) web site was introduced in 2011 and in the five years since the previous publication ongoing research into how the database can better serve forensics has resulted in extensive redesign of the database interface and functionality. Originally designed as a prototype to support forensic use of single nucleotide polymorphisms (SNPs), FROG-kb provides a freely accessible web interface that facilitates forensic practice and can be useful for teaching and research. Based on knowledge gained through its use, the web interface has been redesigned for easier navigation through the multiple components. The site also has functional enhancements, extensive new documentation, and new reference panels of SNPs with new curated data. FROG-kb focuses on single nucleotide polymorphisms (SNPs) and provides reference population data for several published panels of individual identification SNPs (IISNPs) and several published panels of ancestry inference SNPs (AISNPs). For each of the various marker panels with reference population data, FROG-kb calculates random match probabilities (RMP) and relative likelihoods of ancestry for a user-entered genotype profile (either completely or partially specified). Example genotype profiles are available and the User's Manual presents interpretation guidelines for the calculations. The extensive documentation along with ongoing updates makes FROG-kb a comprehensive tool in facilitating use of SNPs in forensic practice and education. An overview of the new FROG-kb with examples and material explaining the results of its use are presented here.