Immunogenetics of HLA-B: SNP, allele, and haplotype diversity in populations from different continents and ancestry backgrounds.

HLA-B is among the most variable gene in the human genome. This gene encodes a key molecule for antigen presentation to CD8+ T lymphocytes and NK cell modulation. Despite the myriad of studies evaluating its coding region (with an emphasis on exons 2 and 3), few studies evaluated introns and regulatory sequences in real population samples. Thus, HLA-B variability is probably underestimated. We applied a bioinformatics pipeline tailored for HLA genes on 5347 samples from 80 different populations, which includes more than 1000 admixed Brazilians, to evaluate the HLA-B variability (SNPs, indels, MNPs, alleles, and haplotypes) in exons, introns, and regulatory regions. We observed 610 variable sites throughout HLA-B; the most frequent variants are shared worldwide. However, the haplotype distribution is geographically structured. We detected 920 full-length haplotypes (exons, introns, and untranslated regions) encoding 239 different protein sequences. HLA-B gene diversity is higher in admixed populations and Europeans while lower in African ancestry individuals. Each HLA-B allele group is associated with specific promoter sequences. This HLA-B variation resource may improve HLA imputation accuracy and disease-association studies and provide evolutionary insights regarding HLA-B genetic diversity in human populations.

Evidence for Epistatic Interaction between HLA-G and LILRB1 in the Pathogenesis of Nonsegmental Vitiligo.

Vitiligo is the most frequent cause of depigmentation worldwide. Genetic association studies have discovered about 50 loci associated with disease, many with immunological functions. Among them is HLA-G, which modulates immunity by interacting with specific inhibitory receptors, mainly LILRB1 and LILRB2. Here we investigated the LILRB1 and LILRB2 association with vitiligo risk and evaluated the possible role of interactions between HLA-G and its receptors in this pathogenesis. We tested the association of the polymorphisms of HLA-G, LILRB1, and LILRB2 with vitiligo using logistic regression along with adjustment by ancestry. Further, methods based on the multifactor dimensionality reduction (MDR) approach (MDR v.3.0.2, GMDR v.0.9, and MB-MDR) were used to detect potential epistatic interactions between polymorphisms from the three genes. An interaction involving rs9380142 and rs2114511 polymorphisms was identified by all methods used. The polymorphism rs9380142 is an HLA-G 3'UTR variant (+3187) with a well-established role in mRNA stability. The polymorphism rs2114511 is located in the exonic region of LILRB1. Although no association involving this SNP has been reported, ChIP-Seq experiments have identified this position as an EBF1 binding site. These results highlight the role of an epistatic interaction between HLA-G and LILRB1 in vitiligo pathogenesis.

Open-Access Worldwide Population STR Database Constructed Using High-Coverage Massively Parallel Sequencing Data Obtained from the 1000 Genomes Project.

Achieving accurate STR genotyping by using next-generation sequencing data has been challenging. To provide the forensic genetics community with a reliable open-access STR database, we conducted a comprehensive genotyping analysis of a set of STRs of broad forensic interest obtained from 1000 Genome populations. We analyzed 22 STR markers using files of the high-coverage dataset of Phase 3 of the 1000 Genomes Project. We used HipSTR to call genotypes from 2504 samples obtained from 26 populations. We were not able to detect the D21S11 marker. The Hardy-Weinberg equilibrium analysis coupled with a comprehensive analysis of allele frequencies revealed that HipSTR was not able to identify longer alleles, which resulted in heterozygote deficiency. Nevertheless, AMOVA, a clustering analysis that uses STRUCTURE, and a Principal Coordinates Analysis showed a clear-cut separation between the four major ancestries sampled by the 1000 Genomes Consortium. Except for larger Penta D and Penta E alleles, and two very small Penta D alleles (2.2 and 3.2) usually observed in African populations, our analyses revealed that allele frequencies and genotypes offered as an open-access database are consistent and reliable.

Genetic diversity of the LILRB1 and LILRB2 coding regions in an admixed Brazilian population sample.

Leukocyte immunoglobulin (Ig)-like receptors (LILR) LILRB1 and LILRB2 may play a pivotal role in maintaining self-tolerance and modulating the immune response through interaction with classical and nonclassical HLA molecules. Although both diversity and natural selection patterns over HLA genes have been extensively evaluated, little information is available concerning the genetic diversity and selection signatures on the LILRB1/2 regions. Therefore, we identified the LILRB1/2 genetic diversity using next-generation sequencing in a population sample from São Paulo State, Brazil. We identified 58 LILRB1 Single Nucleotide Variants (SNVs), which gave rise to 13 haplotypes, and 41 LILRB2 SNVs arranged into 11 haplotypes. Although we may not exclude as a possible effect of population structure, we found evidence of either positive or purifying selection on LILRB1/2 coding regions. Some residues in both proteins showed to be under the effect of positive selection, suggesting that amino acid replacements in these proteins resulted in beneficial functional changes. Finally, we have revealed that allelic variation (six and five amino acid exchanges in LILRB1 and LILRB2, respectively) affects the structure and/or stability of both molecules. Nonetheless, LILRB2 has shown higher average stability, with no D1/D2 residue affecting protein structure. Overall, our findings demonstrate that LILRB1 and LILRB2 are as polymorphic as HLA class Ib genes and provide strong evidence supporting the directional selection regime hypothesis.

Analysis and comparison of the STR genotypes called with HipSTR, STRait Razor and toaSTR by using next generation sequencing data in a Brazilian population sample.

Short tandem repeats (STRs) are particularly difficult to genotype with rapid evolving next-generation sequencing (NGS) technology. Long amplicons containing repetitive sequences result in alignment and genotyping errors. Stutters arising from polymerase slippage often result in reads with additional or missing repeat copies. Many tools are available for analysis of STR markers from NGS data. This study has evaluated the concordance of the HipSTR, STRait Razor, and toaSTR tools for STR genotype calling; NGS data obtained from a highly genetically diverse Brazilian population sample have been used. We found that toaSTR can retrieve a larger number of genotypes (93.8%), whereas HipSTR (84.9%) and STRait Razor present much lower genotype calling (75.3%). Accuracy levels for genotype calling are very similar (identical genotypes ~95% and correct alleles ~ 97.5%) across the three methods. All the markers presenting the same genotype through the methods are in Hardy-Weinberg equilibrium. We found that combined match probability and combined exclusion power are 2.90 × 10-28 and 0.99999999982, respectively. Although toaSTR has varying locus-specific differences and better overall performance of toaSTR, the three programs are reliable genotyping tools. Notwithstanding, additional effort is necessary to improve the genotype calling accuracy of next-generation sequencing datasets.

HLA-G genetic diversity and evolutive aspects in worldwide populations.

HLA-G is a promiscuous immune checkpoint molecule. The HLA-G gene presents substantial nucleotide variability in its regulatory regions. However, it encodes a limited number of proteins compared to classical HLA class I genes. We characterized the HLA-G genetic variability in 4640 individuals from 88 different population samples across the globe by using a state-of-the-art method to characterize polymorphisms and haplotypes from high-coverage next-generation sequencing data. We also provide insights regarding the HLA-G genetic diversity and a resource for future studies evaluating HLA-G polymorphisms in different populations and association studies. Despite the great haplotype variability, we demonstrated that: (1) most of the HLA-G polymorphisms are in introns and regulatory sequences, and these are the sites with evidence of balancing selection, (2) linkage disequilibrium is high throughout the gene, extending up to HLA-A, (3) there are few proteins frequently observed in worldwide populations, with lack of variation in residues associated with major HLA-G biological properties (dimer formation, interaction with leukocyte receptors). These observations corroborate the role of HLA-G as an immune checkpoint molecule rather than as an antigen-presenting molecule. Understanding HLA-G variability across populations is relevant for disease association and functional studies.

Prediction of eye and hair pigmentation phenotypes using the HIrisPlex system in a Brazilian admixed population sample.

Human pigmentation is a complex trait, probably involving more than 100 genes. Predicting phenotypes using SNPs present in those genes is important for forensic purpose. For this, the HIrisPlex tool was developed for eye and hair color prediction, with both models achieving high accuracy among Europeans. Its evaluation in admixed populations is important, since they present a higher frequency of intermediate phenotypes, and HIrisPlex has demonstrated limitations in such predictions; therefore, the performance of this tool may be impaired in such populations. Here, we evaluate the set of 24 markers from the HIrisPlex system in 328 individuals from Ribeirão Preto (SP) region, predicting eye and hair color and comparing the predictions with their real phenotypes. We used the HaloPlex Target Enrichment System and MiSeq Personal Sequencer platform for massively parallel sequencing. The prediction of eye and hair color was accomplished by the HIrisPlex online tool, using the default prediction settings. Ancestry was estimated using the SNPforID 34-plex to observe if and how an individual's ancestry background would affect predictions in this admixed sample. Our sample presented major European ancestry (70.5%), followed by African (21.1%) and Native American/East Asian (8.4%). HIrisPlex presented an overall sensitivity of 0.691 for hair color prediction, with sensitivities ranging from 0.547 to 0.782. The lowest sensitivity was observed for individuals with black hair, who present a reduced European contribution (48.4%). For eye color prediction, the overall sensitivity was 0.741, with sensitivities higher than 0.85 for blue and brown eyes, although it failed in predicting intermediate eye color. Such struggle in predicting this phenotype category is in accordance with what has been seen in previous studies involving HIrisPlex. Individuals with brown eye color are more admixed, with European ancestry decreasing to 62.6%; notwithstanding that, sensitivity for brown eyes was almost 100%. Overall sensitivity increases to 0.791 when a 0.7 threshold is set, though 12.5% of the individuals become undefined. When combining eye and hair prediction, hit rates between 51.3 and 68.9% were achieved. Despite the difficulties with intermediate phenotypes, we have shown that HIrisPlex results can be very helpful when interpreted with caution.

HLA-E gene polymorphisms in chronic hepatitis C: Impact on HLA-E liver expression and disease severity.

Hepatitis C virus usually produces chronic infection and liver damage. Considering that: i) the human leukocyte antigen-E (HLA-E) molecule may modulate the immune response, and ii) little is known about the role of HLA-E gene variability on chronic hepatitis C, we studied the impact of HLA-E polymorphisms on the magnitude of HLA-E liver expression and severity of hepatitis C. HLA-E variability was evaluated in terms of: i) single nucleotide polymorphism (SNP) alleles and genotypes along the gene (beginning of the promoter region, coding region and 3'UTR), and ii) ensemble of SNPs that defines the coding region alleles, considered individually or as genotypes. The comparisons of the HLA-E variation sites between patients and controls revealed no significant results. The HLA-E + 424 T > C (rs1059510), +756 G > A (rs1264457) and + 3777 G > A (rs1059655) variation sites and the HLA-E*01:01:01:01 and HLA-E*01:03:02:01 alleles, considered at single or double doses, were associated with the magnitude of HLA-E liver expression in Kupfer cell, steatosis, inflammatory activity and liver fibrosis. Although these associations were lost after corrections for multiple comparisons, these variable sites may propitiate biological clues for the understanding of the mechanisms associated with hepatitis C severity.

Variability at the 3' untranslated region of the HLA-G gene: a study on patients with AIDS and cytomegalovirus retinochoroiditis.

Cytomegalovirus retinochoroiditis (CMV-R) is the primary cause of blindness among AIDS patients. Since HLA-G is associated with the modulation of the immune response, we hypothesized that variability at the 3' untraslated region (3'UTR) of the gene could be implicated on the predisposition to CMV-R. We evaluated whether HLA-G 3'UTR influences CMV-R development in Brazilian AIDS patients. Peripheral blood DNA was obtained from two groups of patients: (1) AIDS exhibiting CMV-R (n = 40) and (2) AIDS without CMV-R (n = 147). HLA-G 3'UTR typing was performed using sequencing analysis. Allele, genotype and haplotype frequencies were evaluated using Fisher's exact test accompanied by the calculation of the odds ratio and its 95% confidence interval (95% CI). The etiologic (EF) and preventive fractions were also estimated. Compared to AIDS patients without CMV-R, AIDS patients with CMV-R showed increased frequencies of the: (1) + 3001T allele, (2) the + 3001C/T genotype and (3) the UTR-17 (InsTTCCGTGACG) haplotype (EFs = 0.02-0.04). The UTR-3 (DelTCCGCGACG) haplotype was associated with protection against CMV-R development. Although the risk for developing CMR-V at the population level was relatively low (EF), the identification of HLA-G 3'UTR variation sites may help to further evaluate the role of post-transcriptional factors that may contribute to the existent immunosuppresion caused by HIV per se.

Insights on hair, skin and eye color of ancient and contemporary Native Americans.

Over the past few years, tools capable of predicting pigmentation phenotypes have been developed aiming to contribute for criminal and anthropological investigations. In this study, we used eight genetic systems to infer eye, hair, and skin color of ancient and contemporary Native Americans. To achieve this goal, we retrieved 61 SNPs from 42 samples available in free online repositories of DNA sequences. We performed pigmentation predictions using two freely available tools, HIrisPlex-S and Snipper, in addition to two other published models. This workflow made possible to predict all three phenotypes with at least one tool for 29 out of the 42 samples. Considering these 29 individuals, predictions for eye, hair, and skin color were obtained with HIrisPlex-S for 27, 28 and 27 individuals, respectively, while 24, 25 and 25 individuals had such predictions with Snipper. In general, ancient and contemporary Native Americans were predicted to have intermediate/brown eyes, black hair, and intermediate/darker skin pigmentation.

HLA-G liver expression and HLA-G extended haplotypes are associated with chronic hepatitis C in HIV-negative and HIV-coinfected patients.

Chronic hepatitis C virus (HCV) infection induces liver damage and the HCV/Human Immunodeficiency Virus (HIV)-coinfection may further contribute to its progression. The HLA-G molecule inhibits innate and adaptive immunity and may be deleterious for chronically virus-infected cells. Thus we studied 204 HCV-mono-infected patients, 142 HCV/HIV-coinfected patients, 104 HIV-mono-infected patients and 163 healthy subjects. HLA-G liver expression was similarly induced in HCV and HCV/HIV specimens, increasing with advanced fibrosis and necroinflammatory activity, and with increased levels of liver function-related enzymes. Plasma soluble HLA-G (sHLA-G) levels were higher in HCV/HIV patients compared to HCV, HIV and to healthy individuals. sHLA-G continued to be higher in coinfected patients even after stratification of samples according to degree of liver fibrosis and necroinflammatory activity when compared to mono-infected patients. Some HLA-G gene haplotypes differentiated patient groups and presented few associations with liver and plasma HLA-G expression. HLA-G thus may help to distinguish patient groups.

Gene doping: Present and future.

Being an elite athlete is an extremely coveted position, which can lead an individual to use doping. As knowledge is extended, doping techniques have become increasingly sophisticated, and the newest method of doping is gene doping. This article aims to present an updated bibliographic survey that addresses gene doping between 1983 and 2018. Anti-doping agencies have not yet approved any detection technique for this type of doping. The possibility of eradicating such doping is almost zero mainly because gene therapy advances rapidly. In this scenario, the future of gene doping must be discussed and decided before irreversible limits are exceeded.

Applicability of the SNPforID 52-plex panel for human identification and ancestry evaluation in a Brazilian population sample by next-generation sequencing.

SNP analysis is of paramount importance in forensic genetics. The development of new technologies in next-generation sequencing allowed processing a large number of markers in various samples simultaneously. Although SNPs are less informative than STRs, they present lower mutation rates and perform better when using degraded samples. Some SNP systems were developed for forensic usage, such as the SNPforID 52-plex, from the SNPforID Consortium, containing 52 bi-allelic SNPs for human identification. In this paper we evaluated the informativeness of this system in a Brazilian population sample (n = 340). DNA libraries were prepared using a customized HaloPlex Target Enrichment System kit (Agilent Technologies, Inc.) and sequenced in the MiSeq Personal Sequencer platform (Illumina Inc.). The methodology presented here allowed the analysis of 51 out of 52 SNPforID markers. Allele frequencies and forensic parameters were estimated, revealing high informativeness: the combined match probability and power of exclusion were 6.48 × 10-21 and 0.9997, respectively. Population admixture analysis indicates high European contribution (more than 70%) and low Amerindian contribution (less than 10%) in our population, while individual admixture analyses were consistent with the majority of individuals presenting high European contribution. This study demonstrates that the 52-plex kit is suitable for forensic cases in a Brazilian population, presenting results comparable with those obtained using a 16 STR panel.

Comprehensive analysis of HFE gene in hereditary hemochromatosis and in diseases associated with acquired iron overload.

BACKGROUND: Patients with hepatitis C virus (HCV) and hepatocellular carcinoma (HCC) may or not develop iron overload (IO), which is associated with worst prognosis, because can cause serious damage to organs. HFE gene controls the iron uptake from gut, particularly in patients with hereditary hemochromatosis (HH). AIM: To identify associations between HFE coding region in patients exhibiting hereditary hemochromatosis and in diseases associated with acquired IO. METHODS: We sequenced exons 2 to 5 and boundary introns of HFE gene, evaluating all polymorphic sites in patients presenting hereditary (hemochromatosis) or acquired iron overload HCV and HCC) and in healthy controls, using Sanger sequencing. We also determined the ensemble of extended haplotype in healthy control individuals, including several major histocompatibility complex loci, using sequence specific probes. Haplotype reconstruction was performed using the Arlequin and Phase softwares, and linkage disequilibrium (LD) between histocompatibility loci and HFE gene was performed using the Haploview software. RESULTS: The HFE*003 allele was overrepresented (f = 71%) and HFE*001 allele was underrepresented (f = 14%) in HH patients compared to all groups. A strong linkage disequilibrium was observed among the H63D-G, IVS2(+4)-C and C282Y-G gene variants, particularly in HH; however, the mutation IVS2(+4)T>C was not directly associated with HH susceptibility. The HFE*001/HFE*002 genotype conferred susceptibility to HCC in HCV patients exhibiting IO (P = 0.02, OR = 14.14). Although HFE is telomeric to other histocompatibility genes, the H63D-G/IVS2(+4)-C (P ≤ 0.00001/P ≤ 0.0057) combination was in LD with HLA-B*44 allele group in healthy controls. No LD was observed between HFE alleles and other major histocompatibility loci. CONCLUSION: A differential HFE association was observed for HH and for diseases associated with acquired IO (HCV, HCC). Since HFE is very distant from other histocompatibility loci, only weak associations were observed with these alleles.

HLA-C Alleles and Cytomegalovirus Retinitis in Brazilian Patients with AIDS.

PURPOSE: Since cytomegalovirus retinitis (CR) is an important cause of visual impairment among AIDS patients and HLA-C alleles have been associated with AIDS disease outcome, we typed HLA-C locus in patients with AIDS exhibiting or not CR. METHODS: Three groups of individuals were studied: (i) 49 patients with AIDS and CR (Group I), (ii) 161 patients with AIDS without CR (Group II), and (iii) 202 healthy HIV-negative individuals (Group III). HLA-C typing was performed using commercial kits. RESULTS: The HLA-C∗07 allele group was underrepresented in AIDS patients with CR (P=0.005) when compared to controls or when compared to AIDS patients without CR (P=0.006). The HLA-C∗05 allele group was overrepresented in Group II in comparison to Group III (P=0.017). The frequency of the HLA-C∗16 allele group was increased in Group III in comparison to Group II (P=0.004). CONCLUSION: The HLA-C∗07 allele group conferred protection against the development of CR in Brazilian AIDS patients, whereas the HLA-C∗05 and HLA-C∗16 allele groups were associated with AIDS susceptibility or protection, respectively.

Hla-mapper: An application to optimize the mapping of HLA sequences produced by massively parallel sequencing procedures.

A challenging task when more than one HLA gene is evaluated together by second-generation sequencing is to achieve a reliable read mapping. The polymorphic and repetitive nature of HLA genes might bias the read mapping process, usually underestimating variability at very polymorphic segments, or overestimating variability at some segments. To overcome this issue we developed hla-mapper, which takes into account HLA sequences derived from the IPD-IMGT/HLA database and unpublished HLA sequences to apply a scoring system. This comprehends the evaluation of each read pair, addressing them to the most likely HLA gene they were derived from. Hla-mapper provides a reliable map of HLA sequences, allowing accurate downstream analysis such as variant calling, haplotype inference, and allele typing. Moreover, hla-mapper supports whole genome, exome, and targeted sequencing data. To assess the software performance in comparison with traditional mapping algorithms, we used three different simulated datasets to compare the results obtained with hla-mapper, BWA MEM, and Bowtie2. Overall, hla-mapper presented a superior performance, mainly for the classical HLA class I genes, minimizing wrong mapping and cross-mapping that are typically observed when using BWA MEM or Bowtie2 with a single reference genome.

Extended HLA-G genetic diversity and ancestry composition in a Brazilian admixed population sample: Implications for HLA-G transcriptional control and for case-control association studies.

Human leukocyte antigen-G (HLA-G) is a nonclassical Major Histocompatibility Complex (MHC) molecule with immunomodulatory function and restricted tissue expression. The genetic diversity of HLA-G has been extensively studied in several populations, however, the segment located upstream -1406 has not yet been evaluated. We characterized the nucleotide variation and haplotype structure of an extended distal region (-2635), all exons and the 3'UTR segment of HLA-G by next-generation sequencing (NGS) in a sample of 335 Brazilian individuals. We detected 29 variants at the HLA-G distal promoter region, arranged into 19 haplotypes, among which we identified sites that may influence transcription factor targeting. Although the variation pattern in the distal region resembled the one observed in the conventional promoter segment, molecular signature for balancing selection was observed in the promoter segment from -1406 to -1 (Tajima's D = 2.315, P = 0.017), but not in this distal segment (D = 1.049, P = 0.118). Furthermore, the ancestry composition of this Brazilian population sample was determined by the analysis of SNPforID 34-plex ancestry informative marker (AIM) SNP panel. The distribution of HLA-G haplotypes was ancestry-dependent, corroborating previous findings and emphasizing the importance of considering the ancestry information in association studies.

Genetic association between HLA-G 14-bp polymorphism and diseases: A systematic review and meta-analysis.

BACKGROUND: HLA-G is an immune checkpoint molecule. Since a differential molecule expression has been reported even for healthy individuals, many studies have focused on polymorphisms at HLA-G regulatory regions, particularly the 3' untranslated region (3'UTR). The presence/absence of a 14-bp sequence was the first polymorphism described and it is the most studied in association between HLA-G and disorders. METHODS: In this study, we performed a systematic review and meta-analysis of all association studies published regarding the HLA-G 14-bp. RESULTS: We verified association between 14-bp alleles and diseases in the following situations: (1) presence of 14-bp (insertion) conferred susceptibility to preeclampsia (child alleles evaluated) and systemic lupus erythematosus (OR = 1.42; 95%CI = 1.04-1.93; p = 0.026 and OR = 1.13; 95%CI = 1.01-1.27, p = 0.028); (2) 14-bp absence (deletion) was associated with increased risk to breast cancer (OR = 1.23; 95%CI = 1.06-1.43; p = 0.006) and human Cytomegalovirus infection (OR = 2.06; 95%CI = 1.60-2.64; p < 0.0001); and (3) a risk association was observed between the group of reproductive disorders and the 14-bp insertion (OR = 1.12; 95%CI = 1.01-1.24; p = 0.034). CONCLUSIONS: Considering that others 14-bp associations were inconclusive and that other variation sites observed at HLA-G 3'UTR exhibit a proven role on post-transcriptional regulation of HLA-G expression, the complete 3'UTR segment should be analyzed in terms of disease susceptibility, instead of a single polymorphism.

Evaluation of MC1R high-throughput nucleotide sequencing data generated by the 1000 Genomes Project

Abstract The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data.

Evaluation of MC1R high-throughput nucleotide sequencing data generated by the 1000 Genomes Project.

The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data.