An Abstract Interpretation framework for genotype elimination algorithms

We apply Abstract Interpretation to the problem of genotype elimination in pedigrees. First, we give a formalization of some existing algorithms that try to remove from pedigrees all genotypes that violate the Mendelian rules of inheritance. The formalization enables the application of the Abstract Interpretation technique to the problem. We then introduce a particular abstraction, parameterized on given partitions of the set of genotypes. We instantiate this abstraction in order to obtain two existing algorithms for Allele Consolidation, thus giving a formal proof of their correctness. Moreover, the second of these two algorithms is shown to be an example of a forward complete abstraction

Genetic Experiments with Animal Learning: A Critical Review

The basic patterns of inheritance of learning ability in animals have been delineated. Summaries of strain differences in learning rate, responses to selective breeding for learning, heritabilities of learning phenotypes, and heterosis and overdominance are presented. In addition, the patterns of inheritance are shown to vary with the early environment. The causes of genetic differences have received much attention, but much of the research is inconclusive. Both general learning ability and task-specific abilities are important, but their relative importance is not known for most learning tasks. Strain differences have been found to vary widely in response to variations in stimulus parameters, motivational levels, temporal spacing of trials, and pharmacological manipulations. However, in only a few cases have strain differences in learning actually been shown to be attributable to differences in sensory capacities, motivation, memory or activity levels. The physiological bases for differences are totally unknown. The pathways of gene action on learning also await discovery. Although some researchers have claimed to study the adaptive value of learning, their exclusive utilization of laboratory populations precludes meaningful interpretation of their results. Several methodological shortcomings of various experiments are considered, and important areas for future research are suggested

The Influence Of Host Genetics And Different <i>Mycobacterium tuberculosis</i> Strains On Macrophage Functions And Clinical Outcome Of Tuberculosis Disease

Only 5-10 % of Mycobacterium tuberculosis (Mtb) infected individuals develop active pulmonary TB (PTB), and around Mtb isolates are associated with Mtb lineages and distinct host immune responses. I developed assays using ligand coated beads and Mtb reporter strain to measure the macrophage antimicrobial activities. Our assays were able to detect the variation in macrophage activities among different individuals. I investigated the macrophage antimicrobial functions and its association with different TB phenotypes. I measured the macrophage activities in 43 latent TB (LTB) and active TB (ATB) cases combining 54 PTB and 60 TBM patients. Our results showed that macrophages treated with ligands displayed higher antimicrobial activities in LTB than ATB, but no difference between PTB and TBM. Whereas, in Mtb-infected macrophages from both LTB and ATB, proteolysis was reduced due to the modulation of Mtb and there was no difference in their ability to control Mtb. Our data also indicated that the antimicrobial activities of macrophages were ligand-specific or pathway-dependent. The influence of host genetics on TB susceptibility was examined by the association of variants on phagocytic genes using a case-control study with 450 TBM, 450 PTB and 450 controls. Heterozygotes of Macrophage receptor with collagenous structure (MARCO) single nucleotide polymorphisms (SNPs) were associated with increased TB susceptibility, abnormal chest X-ray, infection by Beijing strains and also with impaired macrophage phagocytosis of ligand coated beads. The virulence of Mtb strains was examined by observing cell lysis of macrophages infected with 159 Mtb strains. Virulence phenotype was grouped as low, moderate and high. High virulence was associated with Beijing lineage, reduced TNF-α and IL-6 and increased IL-1β concentration. Overall, this thesis provides new insights into the influence of host and bacterial factors on TB susceptibility. It also provides a foundation for further studies on factors influencing TB susceptibility

Myelin is remodeled cell-autonomously by oligodendroglial macroautophagy

Myelination of axons in the CNS by oligodendrocytes (OLs) is critical for the rapid and reliable conduction of action potentials down neuronal axons, as evidenced by the severe disabilities associated with myelin loss in multiple sclerosis and other diseases of myelin. The specification, differentiation, and maturation of OLs along with myelin formation by OLs have been thoroughly characterized. How myelin is turned over, however remains unclear. It is unsurprising that little is known about myelin turnover considering that for decades following their discovery, myelin and OLs were considered static elements in the adult nervous system. Recent evidence, however, shows that myelin in the CNS is actually plastic. Moreover, myelin remodeling in humans has been suggested to be mediated by mature OLs. As mature OLs have limited capacity to generate new myelin sheaths, we must ask whether mature OLs can remodel the myelin at preexisting myelin sheaths. One intriguing but unproven possibility is that myelin at individual internodes may be remodeled cell-autonomously by mature OLs to modulate neuronal circuit function. Macroautophagy (MA) is responsible for the lysosome-mediated elimination of cytosolic proteins, lipids, and organelles. MA achieves this by capturing cargo in bulk or selectively in a transient, multilamellar structure known as an autophagosome (AP). In this study, we used a combination of in vivo and cellular approaches to test the hypothesis that MA in OLs may be important for myelin remodeling in the adult CNS. We establish that myelin of individual internodes is remodeled, and does so through the coordinated efforts of endocytosis and MA. We found that autophagy protein Atg7 is essential for myelin remodeling in vivo: loss of Atg7 in OLs leads to an age-dependent increase of myelin at the internode and the formation of aberrant myelin structures, most notably myelin outfoldings. In addition, we find that MA has the potential to occur throughout the mature OL, and examination of OLs in culture suggests that formation of a mature AP structure, the amphisome, is required to facilitate the efficient degradation of myelin-containing endocytic structures. Together, we propose that myelin is a dynamic structure that is regularly remodeled through the cooperative efforts of MA and endocytosis. These findings raise the possibility that myelin remodeling is involved in neural plasticity and the tuning of neural circuits

A Drosophila Model Of Sleep Restriction Therapy For Insomnia And Neurodegenerative Disease

Insomnia is the most common sleep disorder among adults, especially affecting individuals of advanced age or with neurodegenerative disease. Humans with insomnia often expand the amount of time they spend in bed in an attempt to compensate for inability to sleep. However, this mismatch of time in bed (high) with sleep ability (low) perpetuates insomnia symptoms. Cognitive Behavioral Therapy for Insomnia (CBT-I) is the first-line insomnia treatment. Sleep restriction – a key component of CBT-I – addresses mismatch between sleep opportunity and ability by restricting time in bed to an amount equal to average sleep ability, leading to enhanced sleep drive and consolidation. Though effective, limited accessibility of practitioners and long duration of therapy are barriers to broad implementation of CBT-I. Deciphering a molecular basis for this behavioral therapy has potential to open new treatment avenues. In Chapter 1, I discuss the utility of modeling insomnia, behavioral therapy, and neurodegenerative disease in Drosophila. In Chapter 2, we develop a Drosophila model for sleep restriction therapy (SRT). We find that restriction of sleep opportunity through manipulation of environmental cues improves sleep efficiency and continuity in multiple short-sleeping Drosophila mutants. We apply SRT to a Drosophila model of Alzheimer’s disease, in which Aβ accumulation causes decreased and fragmented sleep, and demonstrated that sleep restriction reverses these sleep deficits, with associated extension in lifespan. In Chapter 3, we expand our search for fly models of human neurodegenerative disease associated with short-sleeping phenotypes. We find that overexpression of human TDP-43, the protein deposited in intracellular inclusions in ALS and FTD, cause profound sleep disturbances that can be rescued by SRT. TDP-43 flies also exhibit increased arousal threshold and extended longevity with SRT, suggesting deeper sleep with sleep opportunity restriction confers health benefits. In Chapter 4, I discuss ongoing work investigating the intracellular localization of TDP-43, and how improved sleep might mediate toxicity of this protein. Finally, I discuss use of this model to identify molecular signals mediating the response to sleep restriction therapy. These findings have important implications for our understanding of behavioral sleep therapy and its potential as a therapeutic intervention for neurodegenerative disease

Dynamics of Self-Eliminating Gene Drive Mechanisms

Vector-borne diseases continue to be endemic in many parts of the world with dengue, malaria, chikungunya, and yellow fever affecting millions of people every year. Recent advances in genetic engineering, such as CRISPR, have allowed for faster and cheaper DNA modification in organisms with potential to suppress the ability to transmit or carry these pathogens. Additionally, gene drive mechanisms that increase the inheritance rates of transgenic DNA have been proposed, which enable the release of very few transgenic organisms to be capable of transforming entire wild populations. The results of such actions could be irreversible with long-term consequences unknown. Methods to remove transgene DNA have been explored in crops systems and human gene therapy applications. However, such DNA self-elimination mechanisms have not yet been considered to control highly active gene drive transgenes. Here we explore the coupling of three potential gene drive mechanisms (CRISPR, MEDEA, and underdominance) and a proposed self-eliminating mechanism with system dynamics modeling. Our results identify effective parameter spaces for the complete removal of transgenic DNA and restoration of wild-type alleles for all three gene drive mechanisms. Combining gene drive approached with a self-elimination mechanism could allow testing the effects of transgenic populations on the environment, preventing the long-term persistence of the transgene in nature

Using whole-exome sequencing data in an exome-wide association study approach to identify genetic risk factors influencing acute lymphoblastic leukemia response : a focus on asparaginase complications & vincristine-induced peripheral neuropathy

Le traitement de la leucémie lymphoblastique aiguë (LLA) de l’enfant, une affection d'origine maligne des cellules progénitrices lymphoïdes, s’est considérablement amélioré au cours des dernières décennies. En effet, le taux de succès du traitement a dépassé 90% dans des conditions favorables. Cependant, des toxicités liées au traitement peuvent être fatales et entrainer l’interruption ou la cessation du traitement. L'allergie, la pancréatite et la thrombose sont des complications fréquentes du traitement de la LLA et sont associées à l'utilisation de l'asparaginase (ASNase), tandis qu’une toxicité fréquente due à la vincristine (VCR) induit la neuropathie périphérique (VIPN). Étant donné que l’ajustement du schéma posologique afin d’augmenter l'efficacité et diminuer la toxicité est un processus sensible, ceci demeure un défi majeur dans plusieurs protocoles de traitement. La pharmacogénétique étudie comment des altérations de la composante génétique peuvent influer sur la variabilité interindividuelle observée dans la réponse au traitement. Une meilleure compréhension de la base moléculaire de cette variabilité pourrait améliorer considérablement les résultats du traitement, en permettant la personnalisation de ce dernier en fonction du profil génétique du patient. Des études récentes suggèrent l’avantage d’appliquer l’analyse de l’exome à la découverte de variants associés à des traits humains complexes ainsi qu’à des phénotypes de réactions médicamenteuses. L'objectif de notre travail était d'utiliser les données de séquençage pour réaliser des études d'association à l'échelle de l'exome, y compris des étapes de filtrage et de validation, afin d'identifier de nouveaux variants génétiques susceptibles de moduler le risque de développer des complications associées à ASNase et à VIPN. Douze SNP étaient associés à des complications due à l’ASNase dans la cohorte initiale, dont 3 étaient associés à une allergie, 3 à une pancréatite et 6 à une thrombose. Parmi ceux-ci, les variants rs3809849, rs11556218 et rs34708521 des gènes MYBBP1A, IL16 et SPEF2 respectivement ont été associés à des complications multiples et leur association à une pancréatite a été répliquée dans une cohorte de validation indépendante. En ce qui concerne la VCR, trois variantes ont été associées à la modulation du risque de VIPN: rs2781377 dans SYNE2, rs10513762 dans MRPL47 et rs3803357 dans BAHD1. Nous démontrons également le puissant effet combiné de la présence de plusieurs variants de risque pour chacune des toxicités étudiées et fournissons des modèles de prédiction du risque pour la pancréatite et le VIPN basés sur la méthode d’évaluation du risque génétique pondérée et qui ont été validés à l’interne. De plus, étant donné une association du polymorphisme du gène MYBBP1A avec de multiples issus de traitement, nous avons cherché à comprendre comment cette altération génétique se traduit par des variabilités de réponse aux traitements à l’ASNase. En utilisant la technique CRISPR-CAS9 pour induire l'inactivation de gènes dans des lignées cellulaires cancéreuses PANC1 (pancréatiques) nous avons testé la différence de viabilité entre les cellules inactivées et les cellules du type sauvage à la suite de la suppression du gène et du traitement par ASNase. Nos résultats suggèrent un rôle fonctionnel de ce gène dans la modulation de la viabilité, de la capacité de prolifération et de la morphologie des cellules knock-out, ainsi que dans leur sensibilité à l'ASNase, et plaident en outre pour que le gène influence l’issus du traitement de la LLA par ASNase. Le présent travail démontre que l’utilisation de l’approche de séquençage de l’exome entier dans le contexte d’une étude d’association à l’échelle de l’exome est une stratégie valide « sans hypothèse » pour identifier de nouveaux marqueurs génétiques modulant l’effet du traitement de la LLA de l’enfant, et souligne l’importance de l'effet synergique de la combinaison des locus à risque.Treatment of childhood acute lymphoblastic leukemia (ALL), a malignant disorder of lymphoid progenitor cells has improved significantly over the past decades and treatment success rates have surpassed 90% in favorable settings. However, treatment-related toxicities can be life-threatening and cause treatment interruption or cessation. Allergy, pancreatitis and thrombosis are common complications of ALL treatment associated with the use of asparaginase (ASNase), while vincristine-induced peripheral neuropathy (VIPN) is a frequent toxicity of vincristine (VCR). It is a sensitive process and a constant struggle to adjust the dosing regimen to ensure maximum efficacy and minimum toxicity. Pharmacogenetics studies show alterations in the genetic component between individuals can influence the observed variability in treatment response. A better understanding of the molecular basis of this variability in drug effect could significantly improve treatment outcome by allowing the personalization of ALL treatment based on the genetic profile of the patient. Emerging reports suggest the benefit of applying exome analysis to uncover variants associated with complex human traits as well as drug response phenotypes. Our objective in this work was to use available whole-exome sequencing data to perform exome-wide association studies followed by stepwise filtering and validation processes to identify novel variants with a potential to modulate the risk of developing ASNase complications and VIPN. Twelve SNPs were associated with ASNase complications in the discovery cohort including 3 associated with allergy, 3 with pancreatitis and 6 with thrombosis. Of those, rs3809849 in MYBBP1A, rs11556218 in IL16 and rs34708521 in SPEF2 genes were associated with multiple complications and their association with pancreatitis was replicated in an independent validation cohort. As for VCR, three variants were associated with modulating the risk of VIPN: rs2781377 in SYNE2, rs10513762 in MRPL47 and rs3803357 in BAHD1. We also demonstrate a strong combined effect of harbouring multiple risk variants for each of the studied toxicities, and provide internally-validated risk-prediction models based on the weighted genetic risk score method for pancreatitis and VIPN. Furthermore, given the association of the polymorphism in MYBBP1A gene with multiple treatment outcomes, we aimed at understanding how this genetic alteration translates into differences in ASNase treatment response through cell-based functional analysis. Using CRISPR-CAS9 technology we produced gene knockout of PANC1 (pancreatic) cancer cell-lines and tested the difference in viability between the knockouts and wild-type cells following gene deletion and ASNase treatment. Our results suggest a functional role of this gene in modulating the viability, proliferation capacity and the morphology of the knockout cells as well as their sensitivity to ASNase and further advocates the implication of the gene in influencing the outcome of ALL treatment with ASNase. The present work demonstrates that using whole-exome sequencing data in the context of exome-wide association study is a successful “hypothesis-free” strategy for identifying novel genetic markers modulating the effect of childhood ALL treatment and highlights the importance of the synergistic effect of combining risk loci

Genomics and spatial surveillance of Chagas disease and American visceral leishmaniasis

The Trypanosomatidae are a family of parasitic protozoa that infect various animals and plants. Several species within the Trypanosoma and Leishmania genera also pose a major threat to human health. Among these are Trypanosoma cruzi and Leishmania infantum, aetiological agents of the highly debilitating and often deadly vector-borne zoonoses Chagas disease and American visceral leishmaniasis. Current treatment options are far from safe, only partially effective and rarely available in the impoverished regions of Latin America where these ‘neglected tropical diseases’ prevail. Wider-reaching, sustainable protection against T. cruzi and L. infantum might best be achieved by intercepting key routes of zoonotic transmission, but this prophylactic approach requires a better understanding of how these parasites disperse and evolve at various spatiotemporal scales. This dissertation addresses key questions around trypanosomatid parasite biology and spatial epidemiology based on high-resolution, geo-referenced DNA sequence datasets constructed from disease foci throughout Latin America: Which forms of genetic exchange occur in T. cruzi, and are exchange events frequent enough to significantly alter the distribution of important epidemiological traits? How do demographic histories, for example, the recent invasive expansion of L. infantum into the Americas, impact parasite population structure, and do structural changes pose a threat to public health? Can environmental variables predict parasite dispersal patterns at the landscape scale? Following the first chapter’s review of population genetic and genomic approaches in the study of trypanosomatid diseases in Latin America, Chapter 2 describes how reproductive polymorphism segregates T. cruzi populations in southern Ecuador. The study is the first to clearly demonstrate meiotic sex in this species, for decades thought to exchange genetic material only very rarely, and only by non-Mendelian means. T. cruzi subpopulations from the Ecuadorian study site exhibit all major hallmarks of sexual reproduction, including genome-wide Hardy-Weinberg allele frequencies, rapid decay of linkage disequilibrium with map distance and genealogies that fluctuate among chromosomes. The presence of sex promotes the transfer and transformation of genotypes underlying important epidemiological traits, posing great challenges to disease surveillance and the development of diagnostics and drugs. Chapter 3 demonstrates that mating events are also pivotal to L. infantum population structure in Brazil, where introduction bottlenecks have led to striking genetic discontinuities between sympatric strains. Genetic hybridization occurs genome-wide, including at a recently identified ‘miltefosine sensitivity locus’ that appears to be deleted from the majority of Brazilian L. infantum genomes. The study combines an array of genomic and phenotypic analyses to determine whether rapid population expansion or strong purifying selection has driven this prominent > 12 kb deletion to high abundance across Brazil. Results expose deletion size differences that covary with phylogenetic structure and suggest that deletion-carrying strains do not form a private monophyletic clade. These observations are inconsistent with the hypothesis that the deletion genotype rose to high prevalence simply as the result of a founder effect. Enzymatic assays show that loss of ecto-3’-nucleotidase gene function within the deleted locus is coupled to increased ecto-ATPase activity, raising the possibility that alternative metabolic strategies enhance L. infantum fitness in its introduced range. The study also uses demographic simulation modelling to determine whether L. infantum populations in the Americas have expanded from just one or multiple introduction events. Comparison of observed vs. simulated summary statistics using random forests suggests a single introduction from the Old World, but better spatial sampling coverage is required to rule out other demographic scenarios in a pattern-process modelling approach. Further sampling is also necessary to substantiate signs of convergent selection introduced above. Chapter 4 therefore develops a ‘genome-wide locus sequence typing’ (GLST) tool to summarize parasite genetic polymorphism at a fraction of genomic sequencing cost. Applied directly to the infection source (e.g., vector or host tissue), the method also avoids bias from cell purification and culturing steps typically involved prior to sequencing of trypanosomatid and other obligate parasite genomes. GLST scans genomic pilot data for hundreds of polymorphic sequence fragments whose thermodynamic properties permit simultaneous PCR amplification in a single reaction tube. For proof of principle, GLST is applied to metagenomic DNA extracts from various Chagas disease vector species collected in Colombia, Venezuela, and Ecuador. Epimastigote DNA from several T. cruzi reference clones is also analyzed. The method distinguishes 387 single-nucleotide polymorphisms (SNPs) in T. cruzi sub-lineage TcI and an additional 393 SNPs in non-TcI clones. Genetic distances calculated from these SNPs correlate with geographic distances among samples but also distinguish parasites from triatomines collected at common collection sites. The method thereby appears suitable for agent-based spatio-genetic (simulation) analyses left wanted by Chapter 3 – and further formulated in Chapter 5. The potential to survey parasite genetic diversity abundantly across landscapes compels deeper, more systematic exploration of how environmental variables influence the spread of disease. As environmental context is only marginally considered in the population genetic analyses of Chapters 2 – 4, Chapter 5 proposes a new, spatially explicit modelling framework to predict vector-borne parasite gene flow through heterogeneous environment. In this framework, remotely sensed environmental raster values are re-coded and merged into a composite ‘resistance surface’ that summarizes hypothesized effects of landscape features on parasite transmission among vectors and hosts. Parasite population genetic differentiation is then simulated on this surface and fitted to observed diversity patterns in order to evaluate original hypotheses on how environmental variables modulate parasite gene flow. The chapter thereby makes a maiden step from standard population genetic to ‘landscape genomic’ approaches in understanding the ecology and evolution of vector-borne disease. In summary, this dissertation first demonstrates the power of population genetics and genomics to understand fundamental biological properties of important protist parasites, then identifies areas where analytical tools are missing and creates new technical and conceptual frameworks to help fill these gaps. The general discussion (Chapter 6) also outlines several follow-up projects on the key finding of meiotic genetic signatures in T. cruzi. Exploiting recently developed T. cruzi genome-editing systems for the detection of meiotic gene expression and heterozygosis will help understand why and in which life cycle stage some parasite populations use sex and others do not. Long-read sequencing of parental and recombinant genomes will help understand the extent to which sex is diversifying T. cruzi phenotypes, especially virulence and drug resistance properties conferred by surface molecules with repetitive genetic bases intractable to short-read analysis. Chapter 6 also provides follow-up plans for all other research chapters. Emphasis is placed on advancing the complementarity, transferability and public health benefit of the many different methods and concepts employed in this work

Clinical and pathological characteristics of the Uganda genotype of Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the aetiological agent of tuberculosis (TB) is the leading infectious cause of death globally. The outcome of Mtb infection is variable and depends on host, bacterial and environmental factors. Mtb has evolved into a number of lineages and sublineages exhibiting phylogeographical population structuring and diverse clinical consequences after infection. The Mtb Uganda genotype is the commonest cause of pulmonary TB (PTB) in Kampala, Uganda. Prominent inherited host factors that determine the outcome of TB are human leukocyte antigens (HLA). To investigate the impact of Mtb genomic diversity and host HLA allelic variability on the clinical outcome of TB infection in Ugandan patients, the clinical and pathological outcome of Mtb Uganda genotype, and the association between HLA II alleles and PTB due to Mtb were studied. The Uganda genotype was found less frequently in extrapulmonary TB (EPTB) than previously reported in PTB in the same setting (Paper I), and tuberculous lymphadenitis patients infected with Mtb Uganda genotype were significantly less prone to have abdominal lymphadenopathy (Paper IV). This may imply that Mtb Uganda genotype has reduced potential to disseminate. A study of the evolutionary relationships and worldwide distribution of the spoligotypes of Mtb isolates from Ugandan patients with tuberculous lymphadenitis indicated an ongoing evolution of the Uganda genotype, with Uganda at the center of this evolution (Paper II). HIV negative patients with pulmonary TB and their genetically related healthy household controls were typed for HLA class II alleles (Paper III). The HLA- DQB1*03:03 allele was significantly less frequent in patients compared to healthy controls suggesting that the HLADQB1*03:03 allele may be associated with resistance to TB. To establish the cause and pathology of fatal mycobacterial disease, the mycobacteria and pathology associated with fatal TB were studied (Paper IV). One quarter of fatal mycobacterial disease was associated with non tuberculous mycobacteria (NTM). Pleural effusions were significantly associated with Mtb disease compared to NTM infection (Paper IV). To explore the potential use of the CD4+ and CD8+ T cell immunoprofile to diagnose tuberculous effusion, CD4+ and CD8+ T cells from pleural effusions were characterized. CD4+ T cells were significantly more abundant in individuals with TB, and the CD4+/CD8+T cell ratios were significantly higher in tuberculous pleural effusion compared to non tuberculous effusion, however this significance was lost after adjusting for age and ethnicity. Analysis of pleural fluid for the quantity of CD4+ and CD8+ T cells may be useful for establishing a diagnosis of TB in suspicious cases (Paper V). In conclusion, this thesis highlights the genetic diversity of Mtb with Mtb Uganda as the predominant genotype in EPTB patients in Uganda. Both NTM and Mtb are associated with fatal mycobacterial disease and the pathology findings are indistinguishable, though NTM are significantly less likely to cause pleural effusion. Mycobacterial genetic diversity together with host HLA variability may have clinical consequences. This can be exploited in designing TB diagnostic, management and prevention strategies

Culture-free genome-wide locus sequence typing (GLST) provides new perspectives on Trypanosoma cruzi dispersal and infection complexity

El análisis del polimorfismo genético es una poderosa herramienta para la vigilancia epidemiológica y investigar. Sin embargo, la inferencia poderosa de la variación genética del patógeno es a menudo restringido por el acceso limitado al ADN objetivo representativo, especialmente en el estudio de especies parásitas obligadas para las cuales el cultivo ex vivo requiere muchos recursos o es propenso a sesgos. Los métodos modernos de captura de secuencias permiten analizar directamente la variación genética de los patógenos del material del huésped/vector, pero a menudo son demasiado complejos y costosos para entornos de escasos recursos donde prevalecen las enfermedades infecciosas. Este estudio propone un método sencillo y rentable Herramienta de tipificación de secuencias de locus de todo el genoma (GLST) basada en la amplificación paralela masiva de puntos críticos de información en todo el genoma del patógeno objetivo. el multiplexado La reacción en cadena de la polimerasa amplifica cientos de objetivos genéticos diferentes definidos por el usuario en un único tubo de reacción y la posterior limpieza basada en gel de agarosa y código de barras completan la preparación de la biblioteca por menos de 4 USD por muestra. Nuestro estudio genera un modelo flexible Flujo de trabajo de diseño de panel de imprimación GLST para Trypanosoma cruzi, el agente parásito de Chagas enfermedad. Aplicamos con éxito nuestro panel GLST de 203 objetivos a extractos nómicos metagénicos directos y sin cultivo de vectores triatominos que contienen un mínimo de 3,69 pg/μl de ADN de T. cruzi y elaborar más sobre el rendimiento del método mediante la secuenciación de bibliotecas GLST de T. cruzi clones de referencia que representan unidades de tipificación discretas (DTU) TcI, TcIII, TcIV, TcV y TcVI. Los 780 sitios SNP que identificamos en el conjunto de muestras distinguen parásitos de forma repetitiva infectar vectores simpátricos y detectar correlaciones entre distancias genéticas y geográficas a escala regional (< 150 km), así como continental. Los marcadores también separan claramente TcI, TcIII, TcIV y TcV + TcVI y parecen distinguir infecciones multiclonales dentro de TcI. Discutimos las ventajas, limitaciones y perspectivas de nuestro método a través de un espectro de la investigación epidemiológica.Analysis of genetic polymorphism is a powerful tool for epidemiological surveillance and research. Powerful inference from pathogen genetic variation, however, is often restrained by limited access to representative target DNA, especially in the study of obli gate parasitic species for which ex vivo culture is resource-intensive or bias-prone. Mod ern sequence capture methods enable pathogen genetic variation to be analyzed directly from host/vector material but are often too complex and expensive for resource-poor set tings where infectious diseases prevail. This study proposes a simple, cost-effective ‘genome-wide locus sequence typing’ (GLST) tool based on massive parallel amplifica tion of information hotspots throughout the target pathogen genome. The multiplexed polymerase chain reaction amplifies hundreds of different, user-defined genetic targets in a single reaction tube, and subsequent agarose gel-based clean-up and barcoding com pletes library preparation at under 4 USD per sample. Our study generates a flexible GLST primer panel design workflow for Trypanosoma cruzi, the parasitic agent of Chagas disease. We successfully apply our 203-target GLST panel to direct, culture-free metage nomic extracts from triatomine vectors containing a minimum of 3.69 pg/μl T. cruzi DNA and further elaborate on method performance by sequencing GLST libraries from T. cruzi reference clones representing discrete typing units (DTUs) TcI, TcIII, TcIV, TcV and TcVI. The 780 SNP sites we identify in the sample set repeatably distinguish parasites infecting sympatric vectors and detect correlations between genetic and geographic dis tances at regional (< 150 km) as well as continental scales. The markers also clearly sep arate TcI, TcIII, TcIV and TcV + TcVI and appear to distinguish multiclonal infections within TcI. We discuss the advantages, limitations and prospects of our method across a spectrum of epidemiological research