HIV-1 Integrates Widely throughout the Genome of the Human Blood Fluke Schistosoma mansoni.

Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.

Susceptibility to Klebsiella pneumonaie infection in collaborative cross mice is a complex trait controlled by at least three loci acting at different time points.

BACKGROUND: Klebsiella pneumoniae (Kp) is a bacterium causing severe pneumonia in immunocompromised hosts and is often associated with sepsis. With the rise of antibiotic resistant bacteria, there is a need for new effective and affordable control methods; understanding the genetic architecture of susceptibility to Kp will help in their development. We performed the first quantitative trait locus (QTL) mapping study of host susceptibility to Kp infection in immunocompetent Collaborative Cross mice (CC). We challenged 328 mice from 73 CC lines intraperitoneally with 104 colony forming units of Kp strain K2. Survival and body weight were monitored for 15 days post challenge. 48 of the CC lines were genotyped with 170,000 SNPs, with which we mapped QTLs. RESULTS: CC lines differed significantly (P < 0.05) in mean survival time, between 1 to 15 days post infection, and broad sense heritability was 0.45. Distinct QTL were mapped at specific time points during the challenge. A QTL on chromosome 4 was found only on day 2 post infection, and QTL on chromosomes 8 and 18, only on day 8. By using the sequence variations of the eight inbred strain founders of the CC to refine QTL localization we identify several candidate genes. CONCLUSION: Host susceptibility to Kp is a complex trait, controlled by multiple genetic factors that act sequentially during the course of infection.

Pervasive haplotypic variation in the spliceo-transcriptome of the human major histocompatibility complex.

The human major histocompatibility complex (MHC) on chromosome 6p21 is a paradigm for genomics, showing remarkable polymorphism and striking association with immune and non-immune diseases. The complex genomic landscape of the MHC, notably strong linkage disequilibrium, has made resolving causal variants very challenging. A promising approach is to investigate gene expression levels considered as tractable intermediate phenotypes in mapping complex diseases. However, how transcription varies across the MHC, notably relative to specific haplotypes, remains unknown. Here, using an original hybrid tiling and splice junction microarray that includes alternate allele probes, we draw the first high-resolution strand-specific transcription map for three common MHC haplotypes (HLA-A1-B8-Cw7-DR3, HLA-A3-B7-Cw7-DR15, and HLA-A26-B18-Cw5-DR3-DQ2) strongly associated with autoimmune diseases including type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis. We find that haplotype-specific differences in gene expression are common across the MHC, affecting 96 genes (46.4%), most significantly the zing finger protein gene ZFP57. Differentially expressed probes are correlated with polymorphisms between haplotypes, consistent with cis effects that we directly demonstrate for ZFP57 in a cohort of healthy volunteers (P = 1.2 × 10(-14)). We establish that alternative splicing is significantly more frequent in the MHC than genome-wide (72.5% vs. 62.1% of genes, P ≤ 1 × 10(-4)) and shows marked haplotypic differences. We also unmask novel and abundant intergenic transcription involving 31% of transcribed blocks identified. Our study reveals that the renowned MHC polymorphism also manifests as transcript diversity, and our novel haplotype-based approach marks a new step toward identification of regulatory variants involved in the control of MHC-associated phenotypes and diseases.

Collaborative Cross mice and their power to map host susceptibility to Aspergillus fumigatus infection.

The Collaborative Cross (CC) is a genetic reference panel of recombinant inbred lines of mice, designed for the dissection of complex traits and gene networks. Each line is independently descended from eight genetically diverse founder strains such that the genomes of the CC lines, once fully inbred, are fine-grained homozygous mosaics of the founder haplotypes. We present an analysis of 120 CC lines, from a cohort of the CC bred at Tel Aviv University in collaboration with the University of Oxford, which at the time of this study were between the sixth and 12th generations of inbreeding and substantially homozygous at 170,000 SNPs. We show how CC genomes decompose into mosaics, and we identify loci that carry a deficiency or excess of a founder, many being deficient for the wild-derived strains WSB/EiJ and PWK/PhJ. We phenotyped 371 mice from 66 CC lines for a susceptibility to Aspergillus fumigatus infection. The survival time after infection varied significantly between CC lines. Quantitative trait locus (QTL) mapping identified genome-wide significant QTLs on chromosomes 2, 3, 8, 10 (two QTLs), 15, and 18. Simulations show that QTL mapping resolution (the median distance between the QTL peak and true location) varied between 0.47 and 1.18 Mb. Most of the QTLs involved contrasts between wild-derived founder strains and therefore would not segregate between classical inbred strains. Use of variation data from the genomes of the CC founder strains refined these QTLs further and suggested several candidate genes. These results support the use of the CC for dissecting complex traits.

Genetic analysis of complex traits in the emerging Collaborative Cross.

The Collaborative Cross (CC) is a mouse recombinant inbred strain panel that is being developed as a resource for mammalian systems genetics. Here we describe an experiment that uses partially inbred CC lines to evaluate the genetic properties and utility of this emerging resource. Genome-wide analysis of the incipient strains reveals high genetic diversity, balanced allele frequencies, and dense, evenly distributed recombination sites-all ideal qualities for a systems genetics resource. We map discrete, complex, and biomolecular traits and contrast two quantitative trait locus (QTL) mapping approaches. Analysis based on inferred haplotypes improves power, reduces false discovery, and provides information to identify and prioritize candidate genes that is unique to multifounder crosses like the CC. The number of expression QTLs discovered here exceeds all previous efforts at eQTL mapping in mice, and we map local eQTL at 1-Mb resolution. We demonstrate that the genetic diversity of the CC, which derives from random mixing of eight founder strains, results in high phenotypic diversity and enhances our ability to map causative loci underlying complex disease-related traits.

Bioinformatics tools and database resources for systems genetics analysis in mice--a short review and an evaluation of future needs.

During a meeting of the SYSGENET working group 'Bioinformatics', currently available software tools and databases for systems genetics in mice were reviewed and the needs for future developments discussed. The group evaluated interoperability and performed initial feasibility studies. To aid future compatibility of software and exchange of already developed software modules, a strong recommendation was made by the group to integrate HAPPY and R/qtl analysis toolboxes, GeneNetwork and XGAP database platforms, and TIQS and xQTL processing platforms. R should be used as the principal computer language for QTL data analysis in all platforms and a 'cloud' should be used for software dissemination to the community. Furthermore, the working group recommended that all data models and software source code should be made visible in public repositories to allow a coordinated effort on the use of common data structures and file formats.

A Multiparent Advanced Generation Inter-Cross to fine-map quantitative traits in Arabidopsis thaliana.

Identifying natural allelic variation that underlies quantitative trait variation remains a fundamental problem in genetics. Most studies have employed either simple synthetic populations with restricted allelic variation or performed association mapping on a sample of naturally occurring haplotypes. Both of these approaches have some limitations, therefore alternative resources for the genetic dissection of complex traits continue to be sought. Here we describe one such alternative, the Multiparent Advanced Generation Inter-Cross (MAGIC). This approach is expected to improve the precision with which QTL can be mapped, improving the outlook for QTL cloning. Here, we present the first panel of MAGIC lines developed: a set of 527 recombinant inbred lines (RILs) descended from a heterogeneous stock of 19 intermated accessions of the plant Arabidopsis thaliana. These lines and the 19 founders were genotyped with 1,260 single nucleotide polymorphisms and phenotyped for development-related traits. Analytical methods were developed to fine-map quantitative trait loci (QTL) in the MAGIC lines by reconstructing the genome of each line as a mosaic of the founders. We show by simulation that QTL explaining 10% of the phenotypic variance will be detected in most situations with an average mapping error of about 300 kb, and that if the number of lines were doubled the mapping error would be under 200 kb. We also show how the power to detect a QTL and the mapping accuracy vary, depending on QTL location. We demonstrate the utility of this new mapping population by mapping several known QTL with high precision and by finding novel QTL for germination data and bolting time. Our results provide strong support for similar ongoing efforts to produce MAGIC lines in other organisms.

Genomic analysis of natural intra-specific hybrids among Ethiopian isolates of Leishmania donovani.

Parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) cause widespread and devastating human diseases. Visceral leishmaniasis due to Leishmania donovani is endemic in Ethiopia where it has also been responsible for major epidemics. The presence of hybrid genotypes has been widely reported in surveys of natural populations, genetic variation reported in a number of Leishmania species, and the extant capacity for genetic exchange demonstrated in laboratory experiments. However, patterns of recombination and the evolutionary history of admixture that produced these hybrid populations remain unclear. Here, we use whole-genome sequence data to investigate Ethiopian L. donovani isolates previously characterized as hybrids by microsatellite and multi-locus sequencing. To date there is only one previous study on a natural population of Leishmania hybrids based on whole-genome sequences. We propose that these hybrids originate from recombination between two different lineages of Ethiopian L. donovani occurring in the same region. Patterns of inheritance are more complex than previously reported with multiple, apparently independent, origins from similar parents that include backcrossing with parental types. Analysis indicates that hybrids are representative of at least three different histories. Furthermore, isolates were highly polysomic at the level of chromosomes with differences between parasites recovered from a recrudescent infection from a previously treated individual. The results demonstrate that recombination is a significant feature of natural populations and contributes to the growing body of data that shows how recombination, and gene flow, shape natural populations of Leishmania.

Global genome diversity of the Leishmania donovani complex.

Protozoan parasites of the Leishmania donovani complex - L. donovani and L. infantum - cause the fatal disease visceral leishmaniasis. We present the first comprehensive genome-wide global study, with 151 cultured field isolates representing most of the geographical distribution. L. donovani isolates separated into five groups that largely coincide with geographical origin but vary greatly in diversity. In contrast, the majority of L. infantum samples fell into one globally-distributed group with little diversity. This picture is complicated by several hybrid lineages. Identified genetic groups vary in heterozygosity and levels of linkage, suggesting different recombination histories. We characterise chromosome-specific patterns of aneuploidy and identified extensive structural variation, including known and suspected drug resistance loci. This study reveals greater genetic diversity than suggested by geographically-focused studies, provides a resource of genomic variation for future work and sets the scene for a new understanding of the evolution and genetics of the Leishmania donovani complex.

Population genomic evidence that human and animal infections in Africa come from the same populations of Dracunculus medinensis.

BACKGROUND: Guinea worm-Dracunculus medinensis-was historically one of the major parasites of humans and has been known since antiquity. Now, Guinea worm is on the brink of eradication, as efforts to interrupt transmission have reduced the annual burden of disease from millions of infections per year in the 1980s to only 54 human cases reported globally in 2019. Despite the enormous success of eradication efforts to date, one complication has arisen. Over the last few years, hundreds of dogs have been found infected with this previously apparently anthroponotic parasite, almost all in Chad. Moreover, the relative numbers of infections in humans and dogs suggests that dogs are currently the principal reservoir on infection and key to maintaining transmission in that country. PRINCIPAL FINDINGS: In an effort to shed light on this peculiar epidemiology of Guinea worm in Chad, we have sequenced and compared the genomes of worms from dog, human and other animal infections. Confirming previous work with other molecular markers, we show that all of these worms are D. medinensis, and that the same population of worms are causing both infections, can confirm the suspected transmission between host species and detect signs of a population bottleneck due to the eradication efforts. The diversity of worms in Chad appears to exclude the possibility that there were no, or very few, worms present in the country during a 10-year absence of reported cases. CONCLUSIONS: This work reinforces the importance of adequate surveillance of both human and dog populations in the Guinea worm eradication campaign and suggests that control programs aiming to interrupt disease transmission should stay aware of the possible emergence of unusual epidemiology as pathogens approach elimination.

Population genetic analysis of Chadian Guinea worms reveals that human and non-human hosts share common parasite populations.

Following almost 10 years of no reported cases, Guinea worm disease (GWD or dracunculiasis) reemerged in Chad in 2010 with peculiar epidemiological patterns and unprecedented prevalence of infection among non-human hosts, particularly domestic dogs. Since 2014, animal infections with Guinea worms have also been observed in the other three countries with endemic transmission (Ethiopia, Mali, and South Sudan), causing concern and generating interest in the parasites' true taxonomic identity and population genetics. We present the first extensive population genetic data for Guinea worm, investigating mitochondrial and microsatellite variation in adult female worms from both human and non-human hosts in the four endemic countries to elucidate the origins of Chad's current outbreak and possible host-specific differences between parasites. Genetic diversity of Chadian Guinea worms was considerably higher than that of the other three countries, even after controlling for sample size through rarefaction, and demographic analyses are consistent with a large, stable parasite population. Genealogical analyses eliminate the other three countries as possible sources of parasite reintroduction into Chad, and sequence divergence and distribution of genetic variation provide no evidence that parasites in human and non-human hosts are separate species or maintain isolated transmission cycles. Both among and within countries, geographic origin appears to have more influence on parasite population structure than host species. Guinea worm infection in non-human hosts has been occasionally reported throughout the history of the disease, particularly when elimination programs appear to be reaching their end goals. However, no previous reports have evaluated molecular support of the parasite species identity. Our data confirm that Guinea worms collected from non-human hosts in the remaining endemic countries of Africa are Dracunculus medinensis and that the same population of worms infects both humans and dogs in Chad. Our genetic data and the epidemiological evidence suggest that transmission in the Chadian context is currently being maintained by canine hosts.

Gene Expression in Leishmania Is Regulated Predominantly by Gene Dosage.

Leishmania tropica, a unicellular eukaryotic parasite present in North and East Africa, the Middle East, and the Indian subcontinent, has been linked to large outbreaks of cutaneous leishmaniasis in displaced populations in Iraq, Jordan, and Syria. Here, we report the genome sequence of this pathogen and 7,863 identified protein-coding genes, and we show that the majority of clinical isolates possess high levels of allelic diversity, genetic admixture, heterozygosity, and extensive aneuploidy. By utilizing paired genome-wide high-throughput DNA sequencing (DNA-seq) with RNA-seq, we found that gene dosage, at the level of individual genes or chromosomal "somy" (a general term covering disomy, trisomy, tetrasomy, etc.), accounted for greater than 85% of total gene expression variation in genes with a 2-fold or greater change in expression. High gene copy number variation (CNV) among membrane-bound transporters, a class of proteins previously implicated in drug resistance, was found for the most highly differentially expressed genes. Our results suggest that gene dosage is an adaptive trait that confers phenotypic plasticity among natural Leishmania populations by rapid down- or upregulation of transporter proteins to limit the effects of environmental stresses, such as drug selection.IMPORTANCELeishmania is a genus of unicellular eukaryotic parasites that is responsible for a spectrum of human diseases that range from cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL) to life-threatening visceral leishmaniasis (VL). Developmental and strain-specific gene expression is largely thought to be due to mRNA message stability or posttranscriptional regulatory networks for this species, whose genome is organized into polycistronic gene clusters in the absence of promoter-mediated regulation of transcription initiation of nuclear genes. Genetic hybridization has been demonstrated to yield dramatic structural genomic variation, but whether such changes in gene dosage impact gene expression has not been formally investigated. Here we show that the predominant mechanism determining transcript abundance differences (>85%) in Leishmania tropica is that of gene dosage at the level of individual genes or chromosomal somy.

Whole genome resequencing of the human parasite Schistosoma mansoni reveals population history and effects of selection.

Schistosoma mansoni is a parasitic fluke that infects millions of people in the developing world. This study presents the first application of population genomics to S. mansoni based on high-coverage resequencing data from 10 global isolates and an isolate of the closely-related Schistosoma rodhaini, which infects rodents. Using population genetic tests, we document genes under directional and balancing selection in S. mansoni that may facilitate adaptation to the human host. Coalescence modeling reveals the speciation of S. mansoni and S. rodhaini as 107.5-147.6KYA, a period which overlaps with the earliest archaeological evidence for fishing in Africa. Our results indicate that S. mansoni originated in East Africa and experienced a decline in effective population size 20-90KYA, before dispersing across the continent during the Holocene. In addition, we find strong evidence that S. mansoni migrated to the New World with the 16-19th Century Atlantic Slave Trade.

Linkage disequilibrium mapping via cladistic analysis of phase-unknown genotypes and inferred haplotypes in the Genetic Analysis Workshop 14 simulated data.

We recently described a method for linkage disequilibrium (LD) mapping, using cladistic analysis of phased single-nucleotide polymorphism (SNP) haplotypes in a logistic regression framework. However, haplotypes are often not available and cannot be deduced with certainty from the unphased genotypes. One possible two-stage approach is to infer the phase of multilocus genotype data and analyze the resulting haplotypes as if known. Here, haplotypes are inferred using the expectation-maximization (EM) algorithm and the best-guess phase assignment for each individual analyzed. However, inferring haplotypes from phase-unknown data is prone to error and this should be taken into account in the subsequent analysis. An alternative approach is to analyze the phase-unknown multilocus genotypes themselves. Here we present a generalization of the method for phase-known haplotype data to the case of unphased SNP genotypes. Our approach is designed for high-density SNP data, so we opted to analyze the simulated dataset. The marker spacing in the initial screen was too large for our method to be effective, so we used the answers provided to request further data in regions around the disease loci and in null regions. Power to detect the disease loci, accuracy in localizing the true site of the locus, and false-positive error rates are reported for the inferred-haplotype and unphased genotype methods. For this data, analyzing inferred haplotypes outperforms analysis of genotypes. As expected, our results suggest that when there is little or no LD between a disease locus and the flanking region, there will be no chance of detecting it unless the disease variant itself is genotyped.

Evaluation of conjunctival swab sampling in the diagnosis of canine leishmaniasis: A two-year follow-up study in Çukurova Plain, Turkey.

The diagnosis of canine leishmaniasis (CanL) in symptomatic and asymptomatic dogs is a very important and problematic public health issue in Turkey. A longitudinal study was carried out on dogs in selected villages in the Çukurova Plain in Turkey, from July 2011 to June 2013, where cutaneous (CL) and visceral (VL) leishmaniasis is endemic. The study aimed to determine the prevalence of CanL and to evaluate the early diagnostic performance of the non-invasive conjunctival swab nested PCR (CS n-PCR) test in comparison with the Indirect Fluorescent Antibody Test (IFAT). The consecutive blood and CS samples from a representative number of dogs (80-100 dogs/each survey) were collected in a cohort of 6 villages located in the area. Clinical symptoms, demographic and physical features about each dog were noted and lymph node aspiration samples were obtained from selected dogs with lymphadenopathy. In four surveys during the period, a total of 338 sets (blood and CS) of samples from 206 dogs were obtained, such that 83 dogs were sampled more than once. In the cross-sectional analysis, the CanL prevalence was found to be 27.18% (between 7.14% and 39.13%) by IFAT and 41.74% (between 29.03% and 46.66%) by CS n-PCR. The isolated strains were identified as Leishmania infantum MON-1 (n=9) and MON-98 (n=2) by MLEE analysis. Genetic studies targeting the Hsp70 and ITS1 regions performed on 11 dog isolates also showed two clear separate groups. According to IFAT results, 24 of the 83 dogs sampled more than once showed seroconversion (n=19) or a four-fold increase in Ab titers (n=5), while 17 were positive in the initial screening. Forty-two dogs stayed negative during the whole period. The natural Leishmania exposure rate was detected as 31.14% in the study area. CS n-PCR only detected Leishmania infection earlier than IFAT in 8 dogs. No statistical difference was found after the analysis of demographical and physical data. The results indicated that (i) circulation of the dog population is very common in settlements in the Çukurova Plain, but the disease prevalence is high and stable, (ii) the performance of CS n-PCR for detecting Leishmania-dog contact is higher than IFAT, (iii) and some of the parasites isolated from dogs have different zymodemes and/or genotypes from previous human and sand fly isolates; suggesting the probability of two different cycles of leishmaniasis in this particular area. This hypothesis should be supported by future studies targeting vectors and reservoirs.

The effect of genome-wide association scan quality control on imputation outcome for common variants.

Imputation is an extremely valuable tool in conducting and synthesising genome-wide association studies (GWASs). Directly typed SNP quality control (QC) is thought to affect imputation quality. It is, therefore, common practise to use quality-controlled (QCed) data as an input for imputing genotypes. This study aims to determine the effect of commonly applied QC steps on imputation outcomes. We performed several iterations of imputing SNPs across chromosome 22 in a dataset consisting of 3177 samples with Illumina 610 k (Illumina, San Diego, CA, USA) GWAS data, applying different QC steps each time. The imputed genotypes were compared with the directly typed genotypes. In addition, we investigated the correlation between alternatively QCed data. We also applied a series of post-imputation QC steps balancing elimination of poorly imputed SNPs and information loss. We found that the difference between the unQCed data and the fully QCed data on imputation outcome was minimal. Our study shows that imputation of common variants is generally very accurate and robust to GWAS QC, which is not a major factor affecting imputation outcome. A minority of common-frequency SNPs with particular properties cannot be accurately imputed regardless of QC stringency. These findings may not generalise to the imputation of low frequency and rare variants.

Bayesian quantitative trait locus mapping using inferred haplotypes.

We describe a fast hierarchical Bayesian method for mapping quantitative trait loci by haplotype-based association, applicable when haplotypes are not observed directly but are inferred from multiple marker genotypes. The method avoids the use of a Monte Carlo Markov chain by employing priors for which the likelihood factorizes completely. It is parameterized by a single hyperparameter, the fraction of variance explained by the quantitative trait locus, compared to the frequentist fixed-effects model, which requires a parameter for the phenotypic effect of each combination of haplotypes; nevertheless it still provides estimates of haplotype effects. We use simulation to show that the method matches the power of the frequentist regression model and, when the haplotypes are inferred, exceeds it for small QTL effect sizes. The Bayesian estimates of the haplotype effects are more accurate than the frequentist estimates, for both known and inferred haplotypes, which indicates that this advantage is independent of the effect of uncertainty in haplotype inference and will hold in comparison with frequentist methods in general. We apply the method to data from a panel of recombinant inbred lines of Arabidopsis thaliana, descended from 19 inbred founders.

Gene X environment interactions at the serotonin transporter locus.

BACKGROUND: Although it is universally accepted that human disease and behavior depend upon both environmental and genetic variation, a view supported by family and twin studies, examples of environmental interactions with genes identified at the molecular level (G x E) are not so well established. METHODS: We carried out a systematic review and meta-analysis of the serotonin transporter (5-HTTLPR) polymorphic region x stressful life event (SLE) literature and investigated to what extent the main effects reported in this literature are consistent with a number of G x E hypotheses. Our aim was to provide a framework in which to assess the robustness of the claim for the presence of an interaction. RESULTS: The results from our systematic review and meta-analysis indicate that the main effect of 5-HTTLPR genotype and the interaction effect between 5-HTTLPR and SLE on risk of depression are negligible. We found that only a minority of studies report a replication that is qualitatively comparable to that in the original report. CONCLUSIONS: Given reasonable assumptions regarding likely genetic and environmental effect sizes, our simulations indicate that published studies are underpowered. This, together with other aspects of the literature, leads us to suggest that the positive results for the 5-HTTLPR x SLE interactions in logistic regression models are compatible with chance findings.

Efficiency and consistency of haplotype tagging of dense SNP maps in multiple samples.

Haplotype tagging is a means of retaining most of the information in high density marker maps, while reducing genotyping requirements. Estimates of the numbers of tagging SNPs required to cover the human genome have varied widely, ranging from 100,000 to 1,000,000. Tagging has been applied to a number of gene-based datasets but has not been evaluated in contexts reflecting those of genome-wide association studies--large chromosome regions and multiple samples drawn from the same population. We analysed 5000 common markers across a 10 Mb segment of human chromosome 20 in three samples (UK Caucasian, CEPH Caucasian, African American) to evaluate tagging efficiency and consistency. Overall, the results indicate a high degree of efficiency, yielding 3-5-fold savings in Caucasians and 2-3-fold savings in African Americans. These levels varied according to linkage disequilibrium (LD) levels, tagging thresholds and allele frequencies, but in high LD regions they did not vary markedly due to marker density. However, a strong positive relationship between marker density and tagging was observed, relating to the fact that increasing marker density yields greater sequence coverage in high LD, thus requiring more tag SNPs to cover a greater fraction of the genome. Encouragingly, whatever the density employed, a high level of robustness was observed between UK and CEPH samples, as most of the htSNPs selected in one sample were also appropriate as tags in the other.