Origins, evolution, and phenotypic impact of new genes.

Ever since the pre-molecular era, the birth of new genes with novel functions has been considered to be a major contributor to adaptive evolutionary innovation. Here, I review the origin and evolution of new genes and their functions in eukaryotes, an area of research that has made rapid progress in the past decade thanks to the genomics revolution. Indeed, recent work has provided initial whole-genome views of the different types of new genes for a large number of different organisms. The array of mechanisms underlying the origin of new genes is compelling, extending way beyond the traditionally well-studied source of gene duplication. Thus, it was shown that novel genes also regularly arose from messenger RNAs of ancestral genes, protein-coding genes metamorphosed into new RNA genes, genomic parasites were co-opted as new genes, and that both protein and RNA genes were composed from scratch (i.e., from previously nonfunctional sequences). These mechanisms then also contributed to the formation of numerous novel chimeric gene structures. Detailed functional investigations uncovered different evolutionary pathways that led to the emergence of novel functions from these newly minted sequences and, with respect to animals, attributed a potentially important role to one specific tissue--the testis--in the process of gene birth. Remarkably, these studies also demonstrated that novel genes of the various types significantly impacted the evolution of cellular, physiological, morphological, behavioral, and reproductive phenotypic traits. Consequently, it is now firmly established that new genes have indeed been major contributors to the origin of adaptive evolutionary novelties.

Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin.

Pseudomonas fluorescens CHA0 produces several secondary metabolites, e.g., the antibiotics pyoluteorin (Plt) and 2,4-diacetylphloroglucinol (Phl), which are important for the suppression of root diseases caused by soil-borne fungal pathogens. A Tn5 insertion mutant of strain CHA0, CHA625, does not produce Phl, shows enhanced Plt production on malt agar, and has lost part of the ability to suppress black root rot in tobacco plants and take-all in wheat. We used a rapid, two-step cloning-out procedure for isolating the wild-type genes corresponding to those inactivated by the Tn5 insertion in strain CHA625. This cloning method should be widely applicable to bacterial genes tagged with Tn5. The region cloned from P. fluorescens contained three complete open reading frames. The deduced gene products, designated PqqFAB, showed extensive similarities to proteins involved in the biosynthesis of pyrroloquinoline quinone (PQQ) in Klebsiella pneumoniae, Acinetobacter calcoaceticus, and Methylobacterium extorquens. PQQ-negative mutants of strain CHA0 were constructed by gene replacement. They lacked glucose dehydrogenase activity, could not utilize ethanol as a carbon source, and showed a strongly enhanced production of Plt on malt agar. These effects were all reversed by complementation with pqq+ recombinant plasmids. The growth of a pqqF mutant on ethanol and normal Plt production were restored by the addition of 16 nM PQQ. However, the Phl- phenotype of strain CHA625 was due not to the pqq defect but presumably to a secondary mutation. In conclusion, a lack of PQQ markedly stimulates the production of Plt in P. fluorescens.

Blood pressure, cardiac, and renal responses to salt and deoxycorticosterone acetate in mice: role of Renin genes.

Several studies have demonstrated that mice are polymorphic for the number of renin genes, with some inbred strains harboring one gene (Ren-1(c)) and other strains containing two genes (Ren-1(d) and Ren-2). In this study, the effects of 1% salt and deoxycorticosterone acetate (DOCA)/salt were investigated in one- and two-renin gene mice, for elucidation of the role of renin in the modulation of BP, cardiac, and renal responses to salt and DOCA. The results demonstrated that, under baseline conditions, mice with two renin genes exhibited 10-fold higher plasma renin activity, 100-fold higher plasma renin concentrations, elevated BP (which was angiotensin II-dependent), and an increased cardiac weight index, compared with one-renin gene mice (all P < 0.01). The presence of two renin genes markedly increased the BP, cardiac, and renal responses to salt. The number of renin genes also modulated the responses to DOCA/salt. In one-renin gene mice, DOCA/salt induced significant renal and cardiac hypertrophy (P < 0.01) even in the absence of any increase in BP. Treatment with losartan, an angiotensin II AT(1) receptor antagonist, decreased BP in two-renin gene mice but not in one-renin gene mice. However, losartan prevented the development of cardiac hypertrophy in both groups of mice. In conclusion, these data demonstrate that renin genes are important determinants of BP and of the responses to salt and DOCA in mice. The results confirm that the Ren-2 gene, which controls renin production mainly in the submaxillary gland, is physiologically active in mice and is not subject to the usual negative feedback control. Finally, these data provide further evidence that mineralocorticoids promote cardiac hypertrophy even in the absence of BP changes. This hypertrophic process is mediated in part by the activation of angiotensin II AT(1) receptors.

Membrane mu poly(A) signal and 3' flanking sequences function as a transcription terminator for immunoglobulin-encoding genes.

Developmentally regulated mechanisms involving alternative RNA splicing and/or polyadenylation, as well as transcription termination, are implicated in controlling the levels of secreted mu (mu s), membrane mu (mu m) and delta immunoglobulin (Ig) heavy chain mRNAs during B cell differentiation (mu gene encodes the mu heavy chain). Using expression vectors constructed with genomic DNA segments composed of the mu m polyadenylation signal region, we analyzed poly(A) site utilization and termination of transcription in stably transfected myeloma cells and in murine fibroblast L cells. We found that the gene segment containing the mu m poly(A) signals, along with 536 bp of downstream flanking sequence, acted as a transcription terminator in both myeloma cells and L cell fibroblasts. Neither a 141-bp DNA fragment (which directed efficient polyadenylation at the mu m site), nor the 536-bp flanking nucleotide sequence alone, were sufficient to obtain a similar regulation. This shows that the mu m poly(A) region plays a central role in controlling developmentally regulated transcription termination by blocking downstream delta gene expression. Because this gene segment exhibited the same RNA processing and termination activities in fibroblasts, it appears that these processes are not tissue-specific.

NLRC5 exclusively transactivates MHC class I and related genes through a distinctive SXY module.

MHC class II (MHCII) genes are transactivated by the NOD-like receptor (NLR) family member CIITA, which is recruited to SXY enhancers of MHCII promoters via a DNA-binding "enhanceosome" complex. NLRC5, another NLR protein, was recently found to control transcription of MHC class I (MHCI) genes. However, detailed understanding of NLRC5's target gene specificity and mechanism of action remained lacking. We performed ChIP-sequencing experiments to gain comprehensive information on NLRC5-regulated genes. In addition to classical MHCI genes, we exclusively identified novel targets encoding non-classical MHCI molecules having important functions in immunity and tolerance. ChIP-sequencing performed with Rfx5(-/-) cells, which lack the pivotal enhanceosome factor RFX5, demonstrated its strict requirement for NLRC5 recruitment. Accordingly, Rfx5-knockout mice phenocopy Nlrc5 deficiency with respect to defective MHCI expression. Analysis of B cell lines lacking RFX5, RFXAP, or RFXANK further corroborated the importance of the enhanceosome for MHCI expression. Although recruited by common DNA-binding factors, CIITA and NLRC5 exhibit non-redundant functions, shown here using double-deficient Nlrc5(-/-)CIIta(-/-) mice. These paradoxical findings were resolved by using a "de novo" motif-discovery approach showing that the SXY consensus sequence occupied by NLRC5 in vivo diverges significantly from that occupied by CIITA. These sequence differences were sufficient to determine preferential occupation and transactivation by NLRC5 or CIITA, respectively, and the S box was found to be the essential feature conferring NLRC5 specificity. These results broaden our knowledge on the transcriptional activities of NLRC5 and CIITA, revealing their dependence on shared enhanceosome factors but their recruitment to distinct enhancer motifs in vivo. Furthermore, we demonstrated selectivity of NLRC5 for genes encoding MHCI or related proteins, rendering it an attractive target for therapeutic intervention. NLRC5 and CIITA thus emerge as paradigms for a novel class of transcriptional regulators dedicated for transactivating extremely few, phylogenetically related genes.

On homology searches by protein Blast and the characterization of the age of genes

Background: It has been shown in a variety of organisms, including mammals, that genes that appeared recently in evolution, for example orphan genes, evolve faster than older genes. Low functional constraints at the time of origin of novel genes may explain these results. However, this observation has been recently attributed to an artifact caused by the inability of Blast to detect the fastest genes in different eukaryotic genomes. Distinguishing between these two possible explanations would be of great importance for any studies dealing with the taxon distribution of proteins and the origin of novel genes. Results: Here we used simulations of protein sequences to examine the capacity of Blast to detect proteins of diverse evolutionary rates in the different species of an eukaryotic phylogenetic tree that included metazoans, fungi and plants. We simulated the evolution of protein genes with the same evolutionary rates than those observed in functional mammalian genes and with among-site rate heterogeneity. Under these conditions, we found that only a very small percentage of simulated ancestral eukaryotic proteins was affected by the Blast artifact. We show that the good detectability of Blast is due to the heterogeneity of protein evolutionary rates at different sites, since only a small conserved motif in a sequence suffices to detect its homologues. Our results indicate that Blast, at least when applied within eukaryotes, only misses homologues of extremely fast-evolving sequences, which are rare in the mammalian genome, as well as sequences evolving homogeneously or pseudogenes.Conclusion: Although great care should be exercised in the recognition of remote homologues, most functional mammalian genes can be detected in eukaryotic genomes by Blast. That is, the majority of functional mammalian genes are not as fast as for not being detected in other metazoans, fungi or plants, if they had been present in these organisms. Thus, the correlation previously found between age and rate seems not to be due to a pure Blast artifact, at least for mammals. This may have important implications to understand the mechanisms by which novel genes originate.

Differences in the evolutionary history of disease genes affected by dominant or recessive mutations

Background: Global analyses of human disease genes by computational methods have yielded important advances in the understanding of human diseases. Generally these studies have treated the group of disease genes uniformly, thus ignoring the type of disease-causing mutations (dominant or recessive). In this report we present a comprehensive study of the evolutionary history of autosomal disease genes separated by mode of inheritance.Results: We examine differences in protein and coding sequence conservation between dominant and recessive human disease genes. Our analysis shows that disease genes affected by dominant mutations are more conserved than those affected by recessive mutations. This could be a consequence of the fact that recessive mutations remain hidden from selection while heterozygous. Furthermore, we employ functional annotation analysis and investigations into disease severity to support this hypothesis. Conclusion: This study elucidates important differences between dominantly- and recessively-acting disease genes in terms of protein and DNA sequence conservation, paralogy and essentiality. We propose that the division of disease genes by mode of inheritance will enhance both understanding of the disease process and prediction of candidate disease genes in the future.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Alps, genes, and chromosomes: their role in the formation of species in the Sorex araneus group (Mammalia, Insectivora), as inferred from two hybrid zones.

During the Pleistocene glaciations, the Alps were an efficient barrier to gene flow between isolated populations, often leading to allopatric speciation. Afterwards, the Alps strongly influenced the post-glacial recolonization of Europe and represent a major suture zone between differentiated populations. Two hybrid zones in the Swiss and French Alps between genetically and chromosomally well-differentiated species-the Valais shrew, Sorex antinorii, and the common shrew, S. araneus-were studied karyotypically and by analyzing the distribution of seven microsatellite loci. In the center of the Haslital hybrid zone the two species coexist over a distance of 900 m. Hybrid karyotypes, among them the most complex known in Sorex, are rare. F-statistics based on microsatellite data revealed a strong heterozygote deficit only in the center of the zone, due to the sympatric distribution of the two species with little hybridization between them. Structuring within the species (both F(IS) and F(ST)) was low. An hierarchical analysis showed a high level of interspecific differentiation. Results were compared with those previously reported in another hybrid zone located at Les Houches in the French Alps. Genetic structuring within and between species was comparable in both hybrid zones, although chromosomal incompatibilities are more important in Haslital, where a linkage block of the race-specific chromosomes should additionally impede gene flow. Evidence for a more restricted gene flow in Haslital comes from the genetically intermediate hybrid karyotypes, whereas in Les Houches, hybrid karyotypes are genetically identical to individuals of the pure karyotypic races. Genic and chromosomal introgression was observed in Les Houches, but not in Haslital. The possible influence of a river, separating the two species at Les Houches, on gene flow is discussed.

Genes, environment and sex : perspectives from ectothermic vertebrates

RESUMELes modèles classiques sur l'évolution des chromosomes sexuels supposent que des gènes sexe- antagonistes s'accumulent sur les chromosomes sexuels, entraînant ainsi l'apparition d'une région non- recombinante, qui se répand progressivement en favorisant l'accumulation de mutations délétères. En accord avec cette théorie, les chromosomes sexuels que l'on observe aujourd'hui chez les mammifères et les oiseaux sont considérablement différenciés. En revanche, chez la plupart des vertébrés ectothermes, les chromosomes sexuels sont indifférenciés et il existe une impressionnante diversité de mécanismes de détermination du sexe. Au cours de cette thèse, j'ai étudié l'évolution des chromosomes sexuels chez les vertébrés ectothermes, en outre pour mieux comprendre ce contraste avec les vertébrés endothermes. L'hypothèse « high-turnover » postule que les chromosomes sexuels sont remplacés régulièrement à partir d'autosomes afin d'éviter leur dégénérescence. L'hypothèse « fountain-of-youth » propose que la recombinaison entre le chromosome X et le chromosome Y au sein de femelles XY empêche la dégénérescence. Les résultats de ma thèse, basés sur des études théoriques et empiriques, suggèrent que les deux processus peuvent être entraînés par l'environnement et ainsi jouent un rôle important dans l'évolution des chromosomes sexuels chez les vertébrés ectothermes.SUMMARYClassical models of sex-chromosome evolution assume that sexually antagonistic genes accumulate on sex chromosomes leading to a non-recombining region, which progressively expands and favors the accumulation of deleterious mutations. Concordant with this theory, sex chromosomes in extant mammals and birds are considerably differentiated. In most ectothermic vertebrates, such as frogs, however, sex chromosomes are undifferentiated and a striking diversity of sex determination systems is observed. This thesis was aimed to investigate this apparent contrast of sex chromosome evolution between endothermic and ectothermic vertebrates. The "high-turnover" hypothesis holds that sex chromosomes arose regularly from autosomes preventing decay. The "fountain-of-youth" hypothesis posits that sex chromosomes undergo episodic X-Y recombination in sex-reversed XY females, thereby purging ("rejuvenating") the Y chromosome. We suggest that both processes likely played an important role in sex chromosome evolution of ectothermic vertebrates. The literature largely views sex determination as a dichotomous process: individual sex is assumed to be determined either by genetic (genotypic sex determination, GSD) or by environmental factors (environmental sex determination, ESD), most often temperature (temperature sex determination, TSD). We endorsed an alternative view, which sees GSD and TSD as the ends of a continuum. The conservatism of molecular processes among different systems of sex determination strongly supports the continuum view. We proposed to define sex as a threshold trait underlain by a liability factor, and reaction norms allowing modeling interactions between genotypic and temperature effects. We showed that temperature changes (due to e.g., climatic changes or range expansions) are expected to provoke turnovers in sex-determination mechanisms maintaining homomorphic sex chromosomes. The balanced lethal system of crested newts might be the result of such a sex determination turnover, originating from two variants of ancient Y-chromosomes. Observations from a group of tree frogs, on the other hand, supported the 'fountain of youth' hypothesis. We then showed that low rates of sex- reversals in species with GSD might actually be adaptive considering joint effects of deleterious mutation purging and sexually antagonistic selection. Ongoing climatic changes are expected to threaten species with TSD by biasing population sex ratios. In contrast, species with GSD are implicitly assumed immune against such changes, because genetic systems are thought to necessarily produce even sex ratios. We showed that this assumption may be wrong and that sex-ratio biases by climatic changes may represent a previously unrecognized extinction threat for some GSD species.

Polymorphisms in DNA-repair genes in a cohort of prostate cancer patients from different areas in Spain: heterogeneity between populations as a confounding factor in association studies

Background: Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. Objective: To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. Design, Setting, and Participants: A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArrayH NT Cycler. Outcome Measurements and Statistical Analysis: Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. Results and Limitations: We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. Conclusion: Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.

dKDM5/LID regulates H3K4me3 dynamics at the transcription-start site (TSS) of actively transcribed developmental genes

H3K4me3 is a histone modification that accumulates at the transcription-start site (TSS) of active genes and is known to be important for transcription activation. The way in which H3K4me3 is regulated at TSS and the actual molecular basis of its contribution to transcription remain largely unanswered. To address these questions, we have analyzed the contribution of dKDM5/LID, the main H3K4me3 demethylase in Drosophila, to the regulation of the pattern of H3K4me3. ChIP-seq results show that, at developmental genes, dKDM5/LID localizes at TSS and regulates H3K4me3. dKDM5/LID target genes are highly transcribed and enriched in active RNApol II and H3K36me3, suggesting a positive contribution to transcription. Expression-profiling show that, though weakly, dKDM5/LID target genes are significantly downregulated upon dKDM5/LID depletion. Furthermore, dKDM5/LID depletion results in decreased RNApol II occupancy, particularly by the promoter-proximal Pol lloser5 form. Our results also show that ASH2, an evolutionarily conserved factor that locates at TSS and is required for H3K4me3, binds and positively regulates dKDM5/LID target genes. However, dKDM5/LID and ASH2 do not bind simultaneously and recognize different chromatin states, enriched in H3K4me3 and not, respectively. These results indicate that, at developmental genes, dKDM5/LID and ASH2 coordinately regulate H3K4me3 at TSS and that this dynamic regulation contributes to transcription.

Networking of differentially expressed genes in human cancer cells resistant to methotrexate

BACKGROUND: The need for an integrated view of data obtained from high-throughput technologies gave rise to network analyses. These are especially useful to rationalize how external perturbations propagate through the expression of genes. To address this issue in the case of drug resistance, we constructed biological association networks of genes differentially expressed in cell lines resistant to methotrexate (MTX). METHODS: Seven cell lines representative of different types of cancer, including colon cancer (HT29 and Caco2), breast cancer (MCF-7 and MDA-MB-468), pancreatic cancer (MIA PaCa-2), erythroblastic leukemia (K562) and osteosarcoma (Saos-2), were used. The differential expression pattern between sensitive and MTX-resistant cells was determined by whole human genome microarrays and analyzed with the GeneSpring GX software package. Genes deregulated in common between the different cancer cell lines served to generate biological association networks using the Pathway Architect software. RESULTS: Dikkopf homolog-1 (DKK1) is a highly interconnected node in the network generated with genes in common between the two colon cancer cell lines, and functional validations of this target using small interfering RNAs (siRNAs) showed a chemosensitization toward MTX. Members of the UDP-glucuronosyltransferase 1A (UGT1A) family formed a network of genes differentially expressed in the two breast cancer cell lines. siRNA treatment against UGT1A also showed an increase in MTX sensitivity. Eukaryotic translation elongation factor 1 alpha 1 (EEF1A1) was overexpressed among the pancreatic cancer, leukemia and osteosarcoma cell lines, and siRNA treatment against EEF1A1 produced a chemosensitization toward MTX. CONCLUSIONS: Biological association networks identified DKK1, UGT1As and EEF1A1 as important gene nodes in MTX-resistance. Treatments using siRNA technology against these three genes showed chemosensitization toward MTX.

Additive effects of HLA alleles and innate immune genes determine viral outcome in HCV infection.

BACKGROUND: Chronic HCV infection is a leading cause of liver-related morbidity globally. The innate and adaptive immune responses are thought to be important in determining viral outcomes. Polymorphisms associated with the IFNL3 (IL28B) gene are strongly associated with spontaneous clearance and treatment outcomes. OBJECTIVE: This study investigates the importance of HLA genes in the context of genetic variation associated with the innate immune genes IFNL3 and KIR2DS3. DESIGN: We assess the collective influence of HLA and innate immune genes on viral outcomes in an Irish cohort of women (n=319) who had been infected from a single source as well as a more heterogeneous cohort (Swiss Cohort, n=461). In the Irish cohort, a number of HLA alleles are associated with different outcomes, and the impact of IFNL3-linked polymorphisms is profound. RESULTS: Logistic regression was performed on data from the Irish cohort, and indicates that the HLA-A*03 (OR 0.36 (0.15 to 0.89), p=0.027) -B*27 (OR 0.12 (0.03 to 0.45), p=<0.001), -DRB1*01:01 (OR 0.2 (0.07 to 0.61), p=0.005), -DRB1*04:01 (OR 0.31 (0.12 to 0.85, p=0.02) and the CC IFNL3 rs12979860 genotypes (OR 0.1 (0.04 to 0.23), p<0.001) are significantly associated with viral clearance. Furthermore, DQB1*02:01 (OR 4.2 (2.04 to 8.66), p=0.008), KIR2DS3 (OR 4.36 (1.62 to 11.74), p=0.004) and the rs12979860 IFNL3 'T' allele are associated with chronic infection. This study finds no interactive effect between IFNL3 and these Class I and II alleles in relation to viral clearance. There is a clear additive effect, however. Data from the Swiss cohort also confirms independent and additive effects of HLA Class I, II and IFNL3 genes in their prediction of viral outcome. CONCLUSIONS: This data supports a critical role for the adaptive immune response in the control of HCV in concert with the innate immune response.

Mapping Bias Overestimates Reference Allele Frequencies at the HLA Genes in the 1000 Genomes Project Phase I Data.

Next-generation sequencing (NGS) technologies have become the standard for data generation in studies of population genomics, as the 1000 Genomes Project (1000G). However, these techniques are known to be problematic when applied to highly polymorphic genomic regions, such as the human leukocyte antigen (HLA) genes. Because accurate genotype calls and allele frequency estimations are crucial to population genomics analyses, it is important to assess the reliability of NGS data. Here, we evaluate the reliability of genotype calls and allele frequency estimates of the single-nucleotide polymorphisms (SNPs) reported by 1000G (phase I) at five HLA genes (HLA-A, -B, -C, -DRB1, and -DQB1). We take advantage of the availability of HLA Sanger sequencing of 930 of the 1092 1000G samples and use this as a gold standard to benchmark the 1000G data. We document that 18.6% of SNP genotype calls in HLA genes are incorrect and that allele frequencies are estimated with an error greater than ±0.1 at approximately 25% of the SNPs in HLA genes. We found a bias toward overestimation of reference allele frequency for the 1000G data, indicating mapping bias is an important cause of error in frequency estimation in this dataset. We provide a list of sites that have poor allele frequency estimates and discuss the outcomes of including those sites in different kinds of analyses. Because the HLA region is the most polymorphic in the human genome, our results provide insights into the challenges of using of NGS data at other genomic regions of high diversity.