Moara Machado

Origin and dynamics of admixture in Brazilians and its effect on the pattern of deleterious mutations

Significance The EPIGEN Brazil Project is the largest Latin-American initiative to study the genomic diversity of admixed populations and its effect on phenotypes. We studied 6,487 Brazilians from three population-based cohorts with different geographic and demographic backgrounds. We identified ancestry components of these populations at a previously unmatched geographic resolution. We broadened our understanding of the African diaspora, the principal destination of which was Brazil, by revealing an African ancestry component that likely derives from the slave trade from Bantu/eastern African populations. In the context of the current debate about how the pattern of deleterious mutations varies between Africans and Europeans, we use whole-genome data to show that continental admixture is the main and complex determinant of the amount of deleterious genotypes in admixed individuals. While South Americans are underrepresented in human genomic diversity studies, Brazil has been a classical model for population genetics studies on admixture. We present the results of the EPIGEN Brazil Initiative, the most comprehensive up-to-date genomic analysis of any Latin-American population. A population-based genome-wide analysis of 6,487 individuals was performed in the context of worldwide genomic diversity to elucidate how ancestry, kinship, and inbreeding interact in three populations with different histories from the Northeast (African ancestry: 50%), Southeast, and South (both with European ancestry >70%) of Brazil. We showed that ancestry-positive assortative mating permeated Brazilian history. We traced European ancestry in the Southeast/South to a wider European/Middle Eastern region with respect to the Northeast, where ancestry seems restricted to Iberia. By developing an approximate Bayesian computation framework, we infer more recent European immigration to the Southeast/South than to the Northeast. Also, the observed low Native-American ancestry (6–8%) was mostly introduced in different regions of Brazil soon after the European Conquest. We broadened our understanding of the African diaspora, the major destination of which was Brazil, by revealing that Brazilians display two within-Africa ancestry components: one associated with non-Bantu/western Africans (more evident in the Northeast and African Americans) and one associated with Bantu/eastern Africans (more present in the Southeast/South). Furthermore, the whole-genome analysis of 30 individuals (42-fold deep coverage) shows that continental admixture rather than local post-Columbian history is the main and complex determinant of the individual amount of deleterious genotypes.

Phred-Phrap package to analyses tools: a pipeline to facilitate population genetics re-sequencing studies

BackgroundTargeted re-sequencing is one of the most powerful and widely used strategies for population genetics studies because it allows an unbiased screening for variation that is suitable for a wide variety of organisms. Examples of studies that require re-sequencing data are evolutionary inferences, epidemiological studies designed to capture rare polymorphisms responsible for complex traits and screenings for mutations in families and small populations with high incidences of specific genetic diseases. Despite the advent of next-generation sequencing technologies, Sanger sequencing is still the most popular approach in population genetics studies because of the widespread availability of automatic sequencers based on capillary electrophoresis and because it is still less prone to sequencing errors, which is critical in population genetics studies. Two popular software applications for re-sequencing studies are Phred-Phrap-Consed-Polyphred, which performs base calling, alignment, graphical edition and genotype calling and DNAsp, which performs a set of population genetics analyses. These independent tools are the start and end points of basic analyses. In between the use of these tools, there is a set of basic but error-prone tasks to be performed with re-sequencing data.ResultsIn order to assist with these intermediate tasks, we developed a pipeline that facilitates data handling typical of re-sequencing studies. Our pipeline: (1) consolidates different outputs produced by distinct Phred-Phrap-Consed contigs sharing a reference sequence; (2) checks for genotyping inconsistencies; (3) reformats genotyping data produced by Polyphred into a matrix of genotypes with individuals as rows and segregating sites as columns; (4) prepares input files for haplotype inferences using the popular software PHASE; and (5) handles PHASE output files that contain only polymorphic sites to reconstruct the inferred haplotypes including polymorphic and monomorphic sites as required by population genetics software for re-sequencing data such as DNAsp.ConclusionWe tested the pipeline in re-sequencing studies of haploid and diploid data in humans, plants, animals and microorganisms and observed that it allowed a substantial decrease in the time required for sequencing analyses, as well as being a more controlled process that eliminates several classes of error that may occur when handling datasets. The pipeline is also useful for investigators using other tools for sequencing and population genetics analyses.

Deep sequencing of HPV16 genomes: A new high-throughput tool for exploring the carcinogenicity and natural history of HPV16 infection

For unknown reasons, there is huge variability in risk conferred by different HPV types and, remarkably, strong differences even between closely related variant lineages within each type. HPV16 is a uniquely powerful carcinogenic type, causing approximately half of cervical cancer and most other HPV-related cancers. To permit the large-scale study of HPV genome variability and precancer/cancer, starting with HPV16 and cervical cancer, we developed a high-throughput next-generation sequencing (NGS) whole-genome method. We designed a custom HPV16 AmpliSeq™ panel that generated 47 overlapping amplicons covering 99% of the genome sequenced on the Ion Torrent Proton platform. After validating with Sanger, the current “gold standard” of sequencing, in 89 specimens with concordance of 99.9%, we used our NGS method and custom annotation pipeline to sequence 796 HPV16-positive exfoliated cervical cell specimens. The median completion rate per sample was 98.0%. Our method enabled us to discover novel SNPs, large contiguous deletions suggestive of viral integration (OR of 27.3, 95% CI 3.3–222, P=0.002), and the sensitive detection of variant lineage coinfections. This method represents an innovative high-throughput, ultra-deep coverage technique for HPV genomic sequencing, which, in turn, enables the investigation of the role of genetic variation in HPV epidemiology and carcinogenesis.

Population Genetics of GYPB and Association Study between GYPB*S/s Polymorphism and Susceptibility to P. falciparum Infection in the Brazilian Amazon

Background Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil. Methods Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection. Results GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity. Conclusion Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.

Genetic interaction between NAT2, GSTM1, GSTT1, CYP2E1, and environmental factors is associated with adverse reactions to anti-tuberculosis drugs.

BackgroundAdverse drug reactions (ADRs) associated with anti-tuberculosis (anti-TB) drug regimens have considerable impact on anti-TB treatment, potentially leading to unsuccessful outcomes. Nevertheless, the risk factors that play a role in anti-TB drug-induced ADRs are not well established. It is well documented that genetic polymorphisms in drug-metabolizing enzymes (DMEs) result in considerably complex variability in anti-TB drug disposition. In addition, the impact of pharmacogenetic variation on the metabolism of anti-TB drugs may be modifiable by environmental exposure. Thus, an assessment of pharmacogenetic variability combined with biomarkers of environmental exposure may be helpful for demonstrating the effect of the gene-environment interaction on susceptibility to ADRs induced by anti-TB drug therapy.ObjectiveThe aim of the study was to investigate the impact of the interaction between environmental risk factors and pharmacogenetic polymorphisms in four common DMEs — N-acetyltransferase 2 (arylamine N-acetyltransferase) [NAT2], glutathione S-transferase theta 1 [GSTT1], glutathione S-transferase mu 1 [GSTM1], and cytochrome P450 2E1 [CYP2E1] — on commonly reported ADRs to first-line anti-TB drugs in 129 patients receiving homogeneous TB treatment.MethodsTB patients monitored during drug treatment were divided into subgroups according to the presence or absence of ADRs. Additionally, the patients’ clinical and demographic characteristics were collected in order to identify the environmental factors that are potential triggers for ADRs induced by anti-TB drug treatment. Pharmacogenetic variability was determined by gene sequencing, TaqMan® assays, or polymerase chain reaction.ResultsThe findings of this study suggest that the NAT2 slow acetylator haplotype, female sex, and smoking are important determinants of susceptibility to ADRs induced by anti-TB drugs. Patients carrying multiple, but not single, polymorphisms in the NAT2, GSTM1, GSTT1, and CYP2E1 genes were found to have an increased risk of ADRs, as revealed by gene-gene interaction analysis. Moreover, we also identified meaningful gene-environment interaction models that resulted in the highest levels of ADR risk.ConclusionThe study findings provide evidence of the clinical impact of the interaction between pharmacogenetic variability and environmental factors on ADRs induced by anti-TB drug therapy. Predictive pharmacogenetic testing and a comprehensive clinical history would therefore be helpful for identification and careful monitoring of patients at high risk of this complication.

A minimum set of ancestry informative markers for determining admixture proportions in a mixed American population: the Brazilian set

The Brazilian population is considered to be highly admixed. The main contributing ancestral populations were European and African, with Amerindians contributing to a lesser extent. The aims of this study were to provide a resource for determining and quantifying individual continental ancestry using the smallest number of SNPs possible, thus allowing for a cost- and time-efficient strategy for genomic ancestry determination. We identified and validated a minimum set of 192 ancestry informative markers (AIMs) for the genetic ancestry determination of Brazilian populations. These markers were selected on the basis of their distribution throughout the human genome, and their capacity of being genotyped on widely available commercial platforms. We analyzed genotyping data from 6487 individuals belonging to three Brazilian cohorts. Estimates of individual admixture using this 192 AIM panels were highly correlated with estimates using ~370 000 genome-wide SNPs: 91%, 92%, and 74% of, respectively, African, European, and Native American ancestry components. Besides that, 192 AIMs are well distributed among populations from these ancestral continents, allowing greater freedom in future studies with this panel regarding the choice of reference populations. We also observed that genetic ancestry inferred by AIMs provides similar association results to the one obtained using ancestry inferred by genomic data (370 K SNPs) in a simple regression model with rs1426654, related to skin pigmentation, genotypes as dependent variable. In conclusion, these markers can be used to identify and accurately quantify ancestry of Latin Americans or US Hispanics/Latino individuals, in particular in the context of fine-mapping strategies that require the quantification of continental ancestry in thousands of individuals.

Evolutionary Dynamics of the Human NADPH Oxidase Genes CYBB, CYBA, NCF2 and NCF4: Functional Implications

The phagocyte NADPH oxidase catalyzes the reduction of O2 to reactive oxygen species with microbicidal activity. It is composed of two membrane-spanning subunits, gp91-phox and p22-phox (encoded by CYBB and CYBA, respectively), and three cytoplasmic subunits, p40-phox, p47-phox, and p67-phox (encoded by NCF4, NCF1, and NCF2, respectively). Mutations in any of these genes can result in chronic granulomatous disease, a primary immunodeficiency characterized by recurrent infections. Using evolutionary mapping, we determined that episodes of adaptive natural selection have shaped the extracellular portion of gp91-phox during the evolution of mammals, which suggests that this region may have a function in host-pathogen interactions. On the basis of a resequencing analysis of approximately 35 kb of CYBB, CYBA, NCF2, and NCF4 in 102 ethnically diverse individuals (24 of African ancestry, 31 of European ancestry, 24 of Asian/Oceanians, and 23 US Hispanics), we show that the pattern of CYBA diversity is compatible with balancing natural selection, perhaps mediated by catalase-positive pathogens. NCF2 in Asian populations shows a pattern of diversity characterized by a differentiated haplotype structure. Our study provides insight into the role of pathogen-driven natural selection in an innate immune pathway and sheds light on the role of CYBA in endothelial, nonphagocytic NADPH oxidases, which are relevant in the pathogenesis of cardiovascular and other complex diseases.

Population genetics of immune-related multilocus copy number variation in Native Americans

While multiallelic copy number variation (mCNV) loci are a major component of genomic variation, quantifying the individual copy number of a locus and defining genotypes is challenging. Few methods exist to study how mCNV genetic diversity is apportioned within and between populations (i.e. to define the population genetic structure of mCNV). These inferences are critical in populations with a small effective size, such as Amerindians, that may not fit the Hardy–Weinberg model due to inbreeding, assortative mating, population subdivision, natural selection or a combination of these evolutionary factors. We propose a likelihood-based method that simultaneously infers mCNV allele frequencies and the population structure parameter f, which quantifies the departure of homozygosity from the Hardy–Weinberg expectation. This method is implemented in the freely available software CNVice, which also infers individual genotypes using information from both the population and from trios, if available. We studied the population genetics of five immune-related mCNV loci associated with complex diseases (beta-defensins, CCL3L1/CCL4L1, FCGR3A, FCGR3B and FCGR2C) in 12 traditional Native American populations and found that the population structure parameters inferred for these mCNVs are comparable to but lower than those for single nucleotide polymorphisms studied in the same populations.

A graph-based approach for designing extensible pipelines

BackgroundIn bioinformatics, it is important to build extensible and low-maintenance systems that are able to deal with the new tools and data formats that are constantly being developed. The traditional and simplest implementation of pipelines involves hardcoding the execution steps into programs or scripts. This approach can lead to problems when a pipeline is expanding because the incorporation of new tools is often error prone and time consuming. Current approaches to pipeline development such as workflow management systems focus on analysis tasks that are systematically repeated without significant changes in their course of execution, such as genome annotation. However, more dynamism on the pipeline composition is necessary when each execution requires a different combination of steps.ResultsWe propose a graph-based approach to implement extensible and low-maintenance pipelines that is suitable for pipeline applications with multiple functionalities that require different combinations of steps in each execution. Here pipelines are composed automatically by compiling a specialised set of tools on demand, depending on the functionality required, instead of specifying every sequence of tools in advance. We represent the connectivity of pipeline components with a directed graph in which components are the graph edges, their inputs and outputs are the graph nodes, and the paths through the graph are pipelines. To that end, we developed special data structures and a pipeline system algorithm. We demonstrate the applicability of our approach by implementing a format conversion pipeline for the fields of population genetics and genetic epidemiology, but our approach is also helpful in other fields where the use of multiple software is necessary to perform comprehensive analyses, such as gene expression and proteomics analyses. The project code, documentation and the Java executables are available under an open source license at http://code.google.com/p/dynamic-pipeline. The system has been tested on Linux and Windows platforms.ConclusionsOur graph-based approach enables the automatic creation of pipelines by compiling a specialised set of tools on demand, depending on the functionality required. It also allows the implementation of extensible and low-maintenance pipelines and contributes towards consolidating openness and collaboration in bioinformatics systems. It is targeted at pipeline developers and is suited for implementing applications with sequential execution steps and combined functionalities. In the format conversion application, the automatic combination of conversion tools increased both the number of possible conversions available to the user and the extensibility of the system to allow for future updates with new file formats.

EPIGEN-Brazil Initiative resources: a Latin American imputation panel and the Scientific Workflow

EPIGEN-Brazil is one of the largest Latin American initiatives at the interface of human genomics, public health, and computational biology. Here, we present two resources to address two challenges to the global dissemination of precision medicine and the development of the bioinformatics know-how to support it. To address the underrepresentation of non-European individuals in human genome diversity studies, we present the EPIGEN-5M+1KGP imputation panel-the fusion of the public 1000 Genomes Project (1KGP) Phase 3 imputation panel with haplotypes derived from the EPIGEN-5M data set (a product of the genotyping of 4.3 million SNPs in 265 admixed individuals from the EPIGEN-Brazil Initiative). When we imputed a target SNPs data set (6487 admixed individuals genotyped for 2.2 million SNPs from the EPIGEN-Brazil project) with the EPIGEN-5M+1KGP panel, we gained 140,452 more SNPs in total than when using the 1KGP Phase 3 panel alone and 788,873 additional high confidence SNPs (info score ≥ 0.8). Thus, the major effect of the inclusion of the EPIGEN-5M data set in this new imputation panel is not only to gain more SNPs but also to improve the quality of imputation. To address the lack of transparency and reproducibility of bioinformatics protocols, we present a conceptual Scientific Workflow in the form of a website that models the scientific process (by including publications, flowcharts, masterscripts, documents, and bioinformatics protocols), making it accessible and interactive. Its applicability is shown in the context of the development of our EPIGEN-5M+1KGP imputation panel. The Scientific Workflow also serves as a repository of bioinformatics resources.