Pedigree-based estimation of human mobile element retrotransposition rates.

Germline mutation rates in humans have been estimated for a variety of mutation types, including single-nucleotide and large structural variants. Here, we directly measure the germline retrotransposition rate for the three active retrotransposon elements: L1, Alu, and SVA. We used three tools for calling mobile element insertions (MEIs) (MELT, RUFUS, and TranSurVeyor) on blood-derived whole-genome sequence (WGS) data from 599 CEPH individuals, comprising 33 three-generation pedigrees. We identified 26 de novo MEIs in 437 births. The retrotransposition rate estimates for Alu elements, one in 40 births, is roughly half the rate estimated using phylogenetic analyses, a difference in magnitude similar to that observed for single-nucleotide variants. The L1 retrotransposition rate is one in 63 births and is within range of previous estimates (1:20-1:200 births). The SVA retrotransposition rate, one in 63 births, is much higher than the previous estimate of one in 900 births. Our large, three-generation pedigrees allowed us to assess parent-of-origin effects and the timing of insertion events in either gametogenesis or early embryonic development. We find a statistically significant paternal bias in Alu retrotransposition. Our study represents the first in-depth analysis of the rate and dynamics of human retrotransposition from WGS data in three-generation human pedigrees.

Discovery of rare, diagnostic AluYb8/9 elements in diverse human populations.

BACKGROUND: Polymorphic human Alu elements are excellent tools for assessing population structure, and new retrotransposition events can contribute to disease. Next-generation sequencing has greatly increased the potential to discover Alu elements in human populations, and various sequencing and bioinformatics methods have been designed to tackle the problem of detecting these highly repetitive elements. However, current techniques for Alu discovery may miss rare, polymorphic Alu elements. Combining multiple discovery approaches may provide a better profile of the polymorphic Alu mobilome. AluYb8/9 elements have been a focus of our recent studies as they are young subfamilies (~2.3 million years old) that contribute ~30% of recent polymorphic Alu retrotransposition events. Here, we update our ME-Scan methods for detecting Alu elements and apply these methods to discover new insertions in a large set of individuals with diverse ancestral backgrounds. RESULTS: We identified 5,288 putative Alu insertion events, including several hundred novel AluYb8/9 elements from 213 individuals from 18 diverse human populations. Hundreds of these loci were specific to continental populations, and 23 non-reference population-specific loci were validated by PCR. We provide high-quality sequence information for 68 rare AluYb8/9 elements, of which 11 have hallmarks of an active source element. Our subfamily distribution of rare AluYb8/9 elements is consistent with previous datasets, and may be representative of rare loci. We also find that while ME-Scan and low-coverage, whole-genome sequencing (WGS) detect different Alu elements in 41 1000 Genomes individuals, the two methods yield similar population structure results. CONCLUSION: Current in-silico methods for Alu discovery may miss rare, polymorphic Alu elements. Therefore, using multiple techniques can provide a more accurate profile of Alu elements in individuals and populations. We improved our false-negative rate as an indicator of sample quality for future ME-Scan experiments. In conclusion, we demonstrate that ME-Scan is a good supplement for next-generation sequencing methods and is well-suited for population-level analyses.

PADRE: Pedigree-Aware Distant-Relationship Estimation.

Accurate estimation of shared ancestry is an important component of many genetic studies; current prediction tools accurately estimate pairwise genetic relationships up to the ninth degree. Pedigree-aware distant-relationship estimation (PADRE) combines relationship likelihoods generated by estimation of recent shared ancestry (ERSA) with likelihoods from family networks reconstructed by pedigree reconstruction and identification of a maximum unrelated set (PRIMUS), improving the power to detect distant relationships between pedigrees. Using PADRE, we estimated relationships from simulated pedigrees and three extended pedigrees, correctly predicting 20% more fourth- through ninth-degree simulated relationships than when using ERSA alone. By leveraging pedigree information, PADRE can even identify genealogical relationships between individuals who are genetically unrelated. For example, although 95% of 13(th)-degree relatives are genetically unrelated, in simulations, PADRE correctly predicted 50% of 13(th)-degree relationships to within one degree of relatedness. The improvement in prediction accuracy was consistent between simulated and actual pedigrees. We also applied PADRE to the HapMap3 CEU samples and report new cryptic relationships and validation of previously described relationships between families. PADRE greatly expands the range of relationships that can be estimated by using genetic data in pedigrees.

Targeted Capture of Phylogenetically Informative Ves SINE Insertions in Genus Myotis.

Identification of retrotransposon insertions in nonmodel taxa can be technically challenging and costly. This has inhibited progress in understanding retrotransposon insertion dynamics outside of a few well-studied species. To address this problem, we have extended a retrotransposon-based capture and sequence method (ME-Scan [mobile element scanning]) to identify insertions belonging to the Ves family of short interspersed elements (SINEs) across seven species of the bat genus Myotis. We identified between 120,000 and 143,000 SINE insertions in six taxa lacking a draft genome by comparing to the M. lucifugus reference genome. On average, each Ves insertion was sequenced to 129.6 × coverage. When mapped back to the M. lucifugus reference genome, all insertions were confidently assigned within a 10-bp window. Polymorphic Ves insertions were identified in each taxon based on their mapped locations. Using cross-species comparisons and the identified insertion positions, a presence-absence matrix was created for approximately 796,000 insertions. Dollo parsimony analysis of more than 85,000 phylogenetically informative insertions recovered strongly supported, monophyletic clades that correspond with the biogeography of each taxa. This phylogeny is similar to previously published mitochondrial phylogenies, with the exception of the placement of M. vivesi. These results support the utility of our variation on ME-Scan to identify polymorphic retrotransposon insertions in taxa without a reference genome and for large-scale retrotransposon-based phylogenetics.

The heritability of gestational age in a two-million member cohort: implications for spontaneous preterm birth.

Preterm birth (PTB), defined as birth prior to a gestational age (GA) of 37 completed weeks, affects more than 10% of births worldwide. PTB is the leading cause of neonatal mortality and is associated with a broad spectrum of lifelong morbidity in survivors. The etiology of spontaneous PTB (SPTB) is complex and has an important genetic component. Previous studies have compared monozygotic and dizygotic twin mothers and their families to estimate the heritability of SPTB, but these approaches cannot separate the relative contributions of the maternal and the fetal genomes to GA or SPTB. Using the Utah Population Database, we assessed the heritability of GA in more than 2 million post-1945 Utah births, the largest familial GA dataset ever assembled. We estimated a narrow-sense heritability of 13.3% for GA and a broad-sense heritability of 24.5%. A maternal effect (which includes the effect of the maternal genome) accounts for 15.2% of the variance of GA, and the remaining 60.3% is contributed by individual environmental effects. Given the relatively low heritability of GA and SPTB in the general population, multiplex SPTB pedigrees are likely to provide more power for gene detection than will samples of unrelated individuals. Furthermore, nongenetic factors provide important targets for therapeutic intervention.

Shared and unique signals of high-altitude adaptation in geographically distinct Tibetan populations.

Recent studies have used a variety of analytical methods to identify genes targeted by selection in high-altitude populations located throughout the Tibetan Plateau. Despite differences in analytic strategies and sample location, hypoxia-related genes, including EPAS1 and EGLN1, were identified in multiple studies. By applying the same analytic methods to genome-wide SNP information used in our previous study of a Tibetan population (n = 31) from the township of Maduo, located in the northeastern corner of the Qinghai-Tibetan Plateau (4200 m), we have identified common targets of natural selection in a second geographically and linguistically distinct Tibetan population (n = 46) in the Tuo Tuo River township (4500 m). Our analyses provide evidence for natural selection based on iHS and XP-EHH signals in both populations at the p<0.02 significance level for EPAS1, EGLN1, HMOX2, and CYP17A1 and for PKLR, HFE, and HBB and HBG2, which have also been reported in other studies. We highlight differences (i.e., stratification and admixture) in the two distinct Tibetan groups examined here and report selection candidate genes common to both groups. These findings should be considered in the prioritization of selection candidate genes in future genetic studies in Tibet.

Genomic analysis of natural selection and phenotypic variation in high-altitude mongolians.

Deedu (DU) Mongolians, who migrated from the Mongolian steppes to the Qinghai-Tibetan Plateau approximately 500 years ago, are challenged by environmental conditions similar to native Tibetan highlanders. Identification of adaptive genetic factors in this population could provide insight into coordinated physiological responses to this environment. Here we examine genomic and phenotypic variation in this unique population and present the first complete analysis of a Mongolian whole-genome sequence. High-density SNP array data demonstrate that DU Mongolians share genetic ancestry with other Mongolian as well as Tibetan populations, specifically in genomic regions related with adaptation to high altitude. Several selection candidate genes identified in DU Mongolians are shared with other Asian groups (e.g., EDAR), neighboring Tibetan populations (including high-altitude candidates EPAS1, PKLR, and CYP2E1), as well as genes previously hypothesized to be associated with metabolic adaptation (e.g., PPARG). Hemoglobin concentration, a trait associated with high-altitude adaptation in Tibetans, is at an intermediate level in DU Mongolians compared to Tibetans and Han Chinese at comparable altitude. Whole-genome sequence from a DU Mongolian (Tianjiao1) shows that about 2% of the genomic variants, including more than 300 protein-coding changes, are specific to this individual. Our analyses of DU Mongolians and the first Mongolian genome provide valuable insight into genetic adaptation to extreme environments.

Mobile element scanning (ME-Scan) identifies thousands of novel Alu insertions in diverse human populations.

Alu retrotransposons are the most numerous and active mobile elements in humans, causing genetic disease and creating genomic diversity. Mobile element scanning (ME-Scan) enables comprehensive and affordable identification of mobile element insertions (MEI) using targeted high-throughput sequencing of multiplexed MEI junction libraries. In a single experiment, ME-Scan identifies nearly all AluYb8 and AluYb9 elements, with high sensitivity for both rare and common insertions, in 169 individuals of diverse ancestry. ME-Scan detects heterozygous insertions in single individuals with 91% sensitivity. Insertion presence or absence states determined by ME-Scan are 95% concordant with those determined by locus-specific PCR assays. By sampling diverse populations from Africa, South Asia, and Europe, we are able to identify 5799 Alu insertions, including 2524 novel ones, some of which occur in exons. Sub-Saharan populations and a Pygmy group in particular carry numerous intermediate-frequency Alu insertions that are absent in non-African groups. There is a significant dearth of exon-interrupting insertions among common Alu polymorphisms, but the density of singleton Alu insertions is constant across exonic and nonexonic regions. In one case, a validated novel singleton Alu interrupts a protein-coding exon of FAM187B. This implies that exonic Alu insertions are generally deleterious and thus eliminated by natural selection, but not so quickly that they cannot be observed as extremely rare variants.

Mobile element biology: new possibilities with high-throughput sequencing.

Mobile elements comprise more than half of the human genome, but until recently their large-scale detection was time consuming and challenging. With the development of new high-throughput sequencing (HTS) technologies, the complete spectrum of mobile element variation in humans can now be identified and analyzed. Thousands of new mobile element insertions (MEIs) have been discovered, yielding new insights into mobile element biology, evolution, and genomic variation. Here, we review several high-throughput methods, with an emphasis on techniques that specifically target MEIs in humans. We highlight recent applications of these methods in evolutionary studies and in the analysis of somatic alterations in human normal and tumor tissues.

Genetic analysis of ancestry, admixture and selection in Bolivian and Totonac populations of the New World.

BACKGROUND: Populations of the Americas were founded by early migrants from Asia, and some have experienced recent genetic admixture. To better characterize the native and non-native ancestry components in populations from the Americas, we analyzed 815,377 autosomal SNPs, mitochondrial hypervariable segments I and II, and 36 Y-chromosome STRs from 24 Mesoamerican Totonacs and 23 South American Bolivians. RESULTS AND CONCLUSIONS: We analyzed common genomic regions from native Bolivian and Totonac populations to identify 324 highly predictive Native American ancestry informative markers (AIMs). As few as 40-50 of these AIMs perform nearly as well as large panels of random genome-wide SNPs for predicting and estimating Native American ancestry and admixture levels. These AIMs have greater New World vs. Old World specificity than previous AIMs sets. We identify highly-divergent New World SNPs that coincide with high-frequency haplotypes found at similar frequencies in all populations examined, including the HGDP Pima, Maya, Colombian, Karitiana, and Surui American populations. Some of these regions are potential candidates for positive selection. European admixture in the Bolivian sample is approximately 12%, though individual estimates range from 0-48%. We estimate that the admixture occurred ~360-384 years ago. Little evidence of European or African admixture was found in Totonac individuals. Bolivians with pre-Columbian mtDNA and Y-chromosome haplogroups had 5-30% autosomal European ancestry, demonstrating the limitations of Y-chromosome and mtDNA haplogroups and the need for autosomal ancestry informative markers for assessing ancestry in admixed populations.

Metabolic insight into mechanisms of high-altitude adaptation in Tibetans.

Recent studies have identified genes involved in high-altitude adaptation in Tibetans. Genetic variants/haplotypes within regions containing three of these genes (EPAS1, EGLN1, and PPARA) are associated with relatively decreased hemoglobin levels observed in Tibetans at high altitude, providing corroborative evidence for genetic adaptation to this extreme environment. The mechanisms that afford adaptation to high-altitude hypoxia, however, remain unclear. Considering the strong metabolic demands imposed by hypoxia, we hypothesized that a shift in fuel preference to glucose oxidation and glycolysis at the expense of fatty acid oxidation would improve adaptation to decreased oxygen availability. Correlations between serum free fatty acid and lactate concentrations in Tibetan groups living at high altitude and putatively selected haplotypes provide insight into this hypothesis. An EPAS1 haplotype that exhibits a signal of positive selection is significantly associated with increased lactate concentration, the product of anaerobic glycolysis. Furthermore, the putatively advantageous PPARA haplotype is correlated with serum free fatty acid concentrations, suggesting a possible decrease in the activity of fatty acid oxidation. Although further studies are required to assess the molecular mechanisms underlying these patterns, these associations suggest that genetic adaptation to high altitude involves alteration in energy utilization pathways.

Crohn's disease and genetic hitchhiking at IBD5.

Inflammatory bowel disease 5 (IBD5) is a 250 kb haplotype on chromosome 5 that is associated with an increased risk of Crohn's disease in Europeans. The OCTN1 gene is centrally located on IBD5 and encodes a transporter of the antioxidant ergothioneine (ET). The 503F variant of OCTN1 is strongly associated with IBD5 and is a gain-of-function mutation that increases absorption of ET. Although 503F has been implicated as the variant potentially responsible for Crohn's disease susceptibility at IBD5, there is little evidence beyond statistical association to support its role in disease causation. We hypothesize that 503F is a recent adaptation in Europeans that swept to relatively high frequency and that disease association at IBD5 results not from 503F itself, but from one or more nearby hitchhiking variants, in the genes IRF1 or IL5. To test for evidence of recent positive selection on the 503F allele, we employed the iHS statistic, which was significant in the European CEU HapMap population (P=0.0007) and European Human Genome Diversity Panel populations (P≤0.01). To evaluate the hypothesis of disease-variant hitchhiking, we performed haplotype association tests on high-density microarray data in a sample of 1,868 Crohn's disease cases and 5,550 controls. We found that 503F haplotypes with recombination breakpoints between OCTN1 and IRF1 or IL5 were not associated with disease (odds ratio [OR]: 1.05, P=0.21). In contrast, we observed strong disease association for 503F haplotypes with no recombination between these three genes (OR: 1.24, P=2.6×10(-8)), as expected if the sweeping haplotype harbored one or more disease-causing mutations in IRF1 or IL5. To further evaluate these disease-gene candidates, we obtained expression data from lower gastrointestinal biopsies of healthy individuals and Crohn's disease patients. We observed a 72% increase in gene expression of IRF1 among Crohn's disease patients (P=0.0006) and no significant difference in expression of OCTN1. Collectively, these data indicate that the 503F variant has increased in frequency due to recent positive selection and that disease-causing variants in linkage disequilibrium with 503F have hitchhiked to relatively high frequency, thus forming the IBD5 risk haplotype. Finally, our association results and expression data support IRF1 as a strong candidate for Crohn's disease causation.

Maximum-likelihood estimation of recent shared ancestry (ERSA).

Accurate estimation of recent shared ancestry is important for genetics, evolution, medicine, conservation biology, and forensics. Established methods estimate kinship accurately for first-degree through third-degree relatives. We demonstrate that chromosomal segments shared by two individuals due to identity by descent (IBD) provide much additional information about shared ancestry. We developed a maximum-likelihood method for the estimation of recent shared ancestry (ERSA) from the number and lengths of IBD segments derived from high-density SNP or whole-genome sequence data. We used ERSA to estimate relationships from SNP genotypes in 169 individuals from three large, well-defined human pedigrees. ERSA is accurate to within one degree of relationship for 97% of first-degree through fifth-degree relatives and 80% of sixth-degree and seventh-degree relatives. We demonstrate that ERSA's statistical power approaches the maximum theoretical limit imposed by the fact that distant relatives frequently share no DNA through a common ancestor. ERSA greatly expands the range of relationships that can be estimated from genetic data and is implemented in a freely available software package.

Ancestry of the Iban is predominantly Southeast Asian: genetic evidence from autosomal, mitochondrial, and Y chromosomes.

Humans reached present-day Island Southeast Asia (ISEA) in one of the first major human migrations out of Africa. Population movements in the millennia following this initial settlement are thought to have greatly influenced the genetic makeup of current inhabitants, yet the extent attributed to different events is not clear. Recent studies suggest that south-to-north gene flow largely influenced present-day patterns of genetic variation in Southeast Asian populations and that late Pleistocene and early Holocene migrations from Southeast Asia are responsible for a substantial proportion of ISEA ancestry. Archaeological and linguistic evidence suggests that the ancestors of present-day inhabitants came mainly from north-to-south migrations from Taiwan and throughout ISEA approximately 4,000 years ago. We report a large-scale genetic analysis of human variation in the Iban population from the Malaysian state of Sarawak in northwestern Borneo, located in the center of ISEA. Genome-wide single-nucleotide polymorphism (SNP) markers analyzed here suggest that the Iban exhibit greatest genetic similarity to Indonesian and mainland Southeast Asian populations. The most common non-recombining Y (NRY) and mitochondrial (mt) DNA haplogroups present in the Iban are associated with populations of Southeast Asia. We conclude that migrations from Southeast Asia made a large contribution to Iban ancestry, although evidence of potential gene flow from Taiwan is also seen in uniparentally inherited marker data.

Toward a more uniform sampling of human genetic diversity: a survey of worldwide populations by high-density genotyping.

High-throughput genotyping data are useful for making inferences about human evolutionary history. However, the populations sampled to date are unevenly distributed, and some areas (e.g., South and Central Asia) have rarely been sampled in large-scale studies. To assess human genetic variation more evenly, we sampled 296 individuals from 13 worldwide populations that are not covered by previous studies. By combining these samples with a data set from our laboratory and the HapMap II samples, we assembled a final dataset of ~250,000 SNPs in 850 individuals from 40 populations. With more uniform sampling, the estimate of global genetic differentiation (F(ST)) substantially decreases from ~16% with the HapMap II samples to ~11%. A panel of copy number variations typed in the same populations shows patterns of diversity similar to the SNP data, with highest diversity in African populations. This unique sample collection also permits new inferences about human evolutionary history. The comparison of haplotype variation among populations supports a single out-of-Africa migration event and suggests that the founding population of Eurasia may have been relatively large but isolated from Africans for a period of time. We also found a substantial affinity between populations from central Asia (Kyrgyzstani and Mongolian Buryat) and America, suggesting a central Asian contribution to New World founder populations.

Mobile element scanning (ME-Scan) by targeted high-throughput sequencing.

BACKGROUND: Mobile elements (MEs) are diverse, common and dynamic inhabitants of nearly all genomes. ME transposition generates a steady stream of polymorphic genetic markers, deleterious and adaptive mutations, and substrates for further genomic rearrangements. Research on the impacts, population dynamics, and evolution of MEs is constrained by the difficulty of ascertaining rare polymorphic ME insertions that occur against a large background of pre-existing fixed elements and then genotyping them in many individuals. RESULTS: Here we present a novel method for identifying nearly all insertions of a ME subfamily in the whole genomes of multiple individuals and simultaneously genotyping (for presence or absence) those insertions that are variable in the population. We use ME-specific primers to construct DNA libraries that contain the junctions of all ME insertions of the subfamily, with their flanking genomic sequences, from many individuals. Individual-specific "index" sequences are designed into the oligonucleotide adapters used to construct the individual libraries. These libraries are then pooled and sequenced using a ME-specific sequencing primer. Mobile element insertion loci of the target subfamily are uniquely identified by their junction sequence, and all insertion junctions are linked to their individual libraries by the corresponding index sequence. To test this method's feasibility, we apply it to the human AluYb8 and AluYb9 subfamilies. In four individuals, we identified a total of 2,758 AluYb8 and AluYb9 insertions, including nearly all those that are present in the reference genome, as well as 487 that are not. Index counts show the sequenced products from each sample reflect the intended proportions to within 1%. At a sequencing depth of 355,000 paired reads per sample, the sensitivity and specificity of ME-Scan are both approximately 95%. CONCLUSIONS: Mobile Element Scanning (ME-Scan) is an efficient method for quickly genotyping mobile element insertions with very high sensitivity and specificity. In light of recent improvements to high-throughput sequencing technology, it should be possible to employ ME-Scan to genotype insertions of almost any mobile element family in many individuals from any species.

Genetic evidence for high-altitude adaptation in Tibet.

Tibetans have lived at very high altitudes for thousands of years, and they have a distinctive suite of physiological traits that enable them to tolerate environmental hypoxia. These phenotypes are clearly the result of adaptation to this environment, but their genetic basis remains unknown. We report genome-wide scans that reveal positive selection in several regions that contain genes whose products are likely involved in high-altitude adaptation. Positively selected haplotypes of EGLN1 and PPARA were significantly associated with the decreased hemoglobin phenotype that is unique to this highland population. Identification of these genes provides support for previously hypothesized mechanisms of high-altitude adaptation and illuminates the complexity of hypoxia-response pathways in humans.

Limited distribution of a cardiomyopathy-associated variant in India.

Heart failure is a leading cause of death of people in South Asia, and cardiomyopathy is a major cause of heart failure. Myosin binding protein C (MYBPC3) is expressed in the heart muscle, where it regulates the cardiac response to adrenergic stimulation and is important for the structural integrity of the sarcomere. Mutations in the MYBPC3 gene are associated with hypertrophic or dilated cardiomyopathies. A 25-base-pair deletion in intron 32 causes skipping of the downstream exon and is associated with familial cardiomyopathy. To date, this deletion is found primarily in India and South Asia, although it is also found at low frequency in Southeast Asia. In order to better characterise the distribution of this variant, we determined its frequency in 447 individuals from 19 populations, including 10 populations from India and neighbouring populations from Pakistan and Nepal. The deletion frequency is over 8% in some of our Indian samples, and it is not present in any of the populations we sampled outside of India. The differences in the deletion frequencies among populations in India are consistent with patterns of variation previously reported and with patterns we observed among Indian populations based on high-density SNP chip data. Our results indicate that the MYBPC3 deletion is primarily found among Indian populations and that its distribution is consistent with genome-wide patterns of variation in India.

Alu repeats increase local recombination rates.

BACKGROUND: Recombination rates vary widely across the human genome, but little of that variation is correlated with known DNA sequence features. The genome contains more than one million Alu mobile element insertions, and these insertions have been implicated in non-homologous recombination, modulation of DNA methylation, and transcriptional regulation. If individual Alu insertions have even modest effects on local recombination rates, they could collectively have a significant impact on the pattern of linkage disequilibrium in the human genome and on the evolution of the Alu family itself. RESULTS: We carried out sequencing, SNP identification, and SNP genotyping around 19 AluY insertion loci in 347 individuals sampled from diverse populations, then used the SNP genotypes to estimate local recombination rates around the AluY loci. The loci and SNPs were chosen so as to minimize other factors (such as SNP ascertainment bias and SNP density) that could influence recombination rate estimates. We detected a significant increase in recombination rate within approximately 2 kb of the AluY insertions in our African population sample. To test this observation against a larger set of AluY insertions, we applied our locus- and SNP-selection design and analyses to the HapMap Phase II data. In that data set, we observed a significantly increased recombination rate near AluY insertions in both the CEU and YRI populations. CONCLUSION: We show that the presence of a fixed AluY insertion is significantly predictive of an elevated local recombination rate within 2 kb of the insertion, independent of other known predictors. The magnitude of this effect, approximately a 6% increase, is comparable to the effects of some recombinogenic DNA sequence motifs identified via their association with recombination hot spots.

Fine-scaled human genetic structure revealed by SNP microarrays.

We report an analysis of more than 240,000 loci genotyped using the Affymetrix SNP microarray in 554 individuals from 27 worldwide populations in Africa, Asia, and Europe. To provide a more extensive and complete sampling of human genetic variation, we have included caste and tribal samples from two states in South India, Daghestanis from eastern Europe, and the Iban from Malaysia. Consistent with observations made by Charles Darwin, our results highlight shared variation among human populations and demonstrate that much genetic variation is geographically continuous. At the same time, principal components analyses reveal discernible genetic differentiation among almost all identified populations in our sample, and in most cases, individuals can be clearly assigned to defined populations on the basis of SNP genotypes. All individuals are accurately classified into continental groups using a model-based clustering algorithm, but between closely related populations, genetic and self-classifications conflict for some individuals. The 250K data permitted high-level resolution of genetic variation among Indian caste and tribal populations and between highland and lowland Daghestani populations. In particular, upper-caste individuals from Tamil Nadu and Andhra Pradesh form one defined group, lower-caste individuals from these two states form another, and the tribal Irula samples form a third. Our results emphasize the correlation of genetic and geographic distances and highlight other elements, including social factors that have contributed to population structure.