1,721,188 research outputs found
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Choosing Controls Wisely to Improve Power in Infectious Disease Genome-Wide Association Studies
No full textThis thesis addresses a crucial aspect of the design of infectious disease genome-wide association studies (GWAS's): the selection of a control group. In particular, we examined how the level of pathogen exposure within the control group and the level of disease susceptibility in the studied population impacts GWAS power to detect genotype-phenotype associations. We developed a simulation that models an infectious disease GWAS and provides power estimates given control exposure, disease susceptibility, and sample sizes. Simulation data demonstrated that control exposure significantly impacts study power and that ideal GWAS designs will have low disease susceptibility and high control exposure. We also found that GWAS's studying highly susceptible populations are particularly vulnerable to power reductions caused by low control exposure, and recommendations were made for the designs of prospective GWAS's using these results. This research presents a general framework for evaluating GWAS power with respect to control exposure and disease susceptibility, as well as a tool that provides quantitative power estimates to help researchers select an effective control cohort and improve their overall GWAS designs
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Evolutionary Adaptation and Antimalarial Resistance in Plasmodium falciparum
Full text linkThe malaria parasite, Plasmodium falciparum, has a demonstrated history of adaptation to antimalarials and host immune pressure. This ability unraveled global eradication programs fifty years ago and seriously threatens renewed efforts today. Despite the magnitude of the global health problem, little is known about the genetic mechanisms by which the parasite evades control efforts. Population genomic methods provide a new way to identify the mutations and genes responsible for drug resistance and other clinically important traits
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Rapid Response, High-Throughput Extraction of Biosafety Level 4 Viral Nucleic Acids
No full textRecent outbreaks of Ebola and Lassa virus have highlighted the need for real-time monitoring of virus evolution and spread using genome sequencing to enable rapid response efforts. For these category A pathogens, this effort requires development of well vetted protocols that assure complete virus inactivation while preserving the quality of nucleic acids. Such methods will allow for safe and effective high-throughput sequencing work outside of biosafety level 4 (BSL-4) facilities, and can ease the burden of manual sample processing, which can create bottlenecks during outbreaks.
The currently preferred methods of viral nucleic acid inactivation and extraction are low to medium throughput at best, involving manual manipulation through every step of the process. In response, I optimized an automated protocol using the KingFisher Flex Magnetic Particle Processor for high-throughput nucleic acid extractions with MagMAXTM bead-based kits, and compare its RNA yield and sequencing coverage against the commonly used Qiagen spin column kit. I also automated many aspects of the extraction and library construction protocol using the Bravo Liquid Handler in order to further increase throughput. One of the most repeated and time-consuming steps in the library construction process is nucleic acid purification using SPRI beads, I thoroughly tested a protocol to automate high-throughput nucleic acid purification on the Bravo Liquid Handler.
I found comparable yields from extractions performed by the KingFisher Flex Magnetic Particle Processor using the MagMax Pathogen Kit and Qiagen spin column, in terms of RNA yield and sequencing coverage, as quantified by RT-qPCR and Illumina next-generation sequencing coverage. I also saw comparable results between high-throughput automated and manual nucleic acid purification assays. Together, I have developed an effective high-throughput protocol to inactivate and process clinical samples that can enable real-time genome sequencing during viral outbreaks.Viral Outbreak; Rapid Response; Infectious Diseases; Ebola; Ebola Virus; Ebola Virus Disease; EBOV; EVD; LASV; Lassa Virus; Lassa Virus Disease; Lassa; Nigeria; Sequencing; Next Generation Sequencing; EV; CMV, Cytomegalovirus; Enterovirus; Viral Inactivation; Disease Outbreak; Disease Detectives; MagMax Pathogen RNA/DNA; RNA extraction
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Metabolic Adaptations in Modern Human Populations: Evidence, Theory, and Investigation
No full textDiverse climates, infectious agents, and subsistence patterns drove humans to adapt metabolically to different environments since the migration out of Africa 100,000 years ago. In this dissertation, I review current literature on the genetic underpinnings, and the molecular and physiological manifestations of these metabolic adaptations in diverse human populations. Then, I develop a theory regarding pregnancy as a critical period in life history that mediated recent selection on human metabolism. Finally, I investigate the function and evidence for selection of derived genetic variants at increased frequency in East Asian populations. I find multiple standing variants that increase expression of the gene IVD and increase the efficiency of leucine catabolism, which lie on positively selected haplotypes in East Asians. I use this research process as a model for how to develop and study novel hypotheses of human metabolic adaptation. Such adaptations often impact health in the modern environment, so more evolutionary research will provide useful guidance to the medical community in how to treat people from diverse ethnicities.Human Evolutionary Biologyhuman evolution; genetics; selection; metabolism; pregnancy; leucin
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Epigenetic Mechanisms in Development and Disease
No full textThis thesis presents three ongoing research projects that each attempt to understand the earliest stages of ontogeny, the assembly of a complex multicellular organism from a single totipotent cell, and how cellular diversity arises given that these cells must interpret an identical genetic template. In chapter one, I focus on the global redistribution of cytosine methylation that occurs within the implantation-stage mouse embryo to create highly divergent embryonic and extraembryonic epigenomic landscapes. Notably, the extraembryonic landscape is characterized by globally reduced DNA methylation levels and non-canonical targeting to promoters of developmental genes, which are generally assumed to be regulated by the Polycomb Repressor Complexes (PRC) 1 and 2 within embryonic lineages but are aberrantly methylated in most cancers. By exploring the epigenetic response to certain growth factors and generating mutant embryos, I establish that this landscape can be acquired deterministically during a brief developmental window and appears to represent the downstream effects of a dedicated pathway, suggesting that this pervasively observed feature in cancer may represent a misappropriated, but developmentally-encoded, mode of genome regulation. In chapter two, I optimize a platform for evaluating mutant mouse embryos using single cell transcriptomics that can be used to investigate the roles of promiscuously utilized epigenetic regulators, which have no innate sequence specificity yet regulate a highly cell-type specific target spectrum. I then present the application of this pipeline to investigate PRC2 as it functions in early lineage commitment. Embryos lacking the essential component Eed fail to properly apportion the primitive streak, leading to a general posteriorization of the mesoderm and overproduction primordial germ cell-like cells. Notably, these defects can be detected within progenitor states before the mutant phenotype is morphologically apparent. Finally, chapter three represents ongoing efforts to develop methods for recording cellular lineage agnostically and at single cell resolution using continuous, stochastic Cas9-based genome editing in lieu of traditional methods. With this tool, my collaborators and I hope to open up new quantitative avenues for studying the robust nature of development, where differentiating cellular fields must be synchronously regulated at the organismal level to effectively reproduce a complete body plan.Biology, Molecular and Cellula
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Global Signatures of Selection in Humans
No full textThis thesis studies the properties and prevalence of natural selection operating on our species on a global or genome-wide scale. It is still not known how negative selection against deleterious mutations operates genome-wide, whether balancing selection is responsible for maintaining higher genetic diversity in some parts of the genome, and how prevalent adaptation on complex or polygenic traits is in humans. We now have access to population genetic data from healthy populations worldwide, methods to evaluate the functional effects and age of genetic mutations, and Genome-wide Association (GWA) studies to estimate the genetic basis of a complex trait.
Using these resources, as well as developing novel statistical methodologies, we show that 1) negative selection in humans involves synergistic epistasis, or deleterious mutations in the human genome interact globally in a manner to reinforce each other’s effects, 2) genes with monoallelic expression contribute disproportionately to genetic diversity in humans, which is maintained through the evolutionary forces of balancing selection and a higher mutation and recombination rate 3) the signal for polygenic adaptation at height-associated genetic variants in humans is confounded by ascertainment biases in the GWAS used to estimate height across populations.
We conclude that (1) helps explain how humans tolerate a high incoming rate of deleterious mutations, and why sexual reproduction may have an evolutionary advantage, (2) establishes a link between genetic and epigenetic mechanisms of maintaining and exhibiting high genetic diversity in humans, and (3) demonstrates the presence of confounders that restrict interpretation of signals of polygenic adaptation in humans found using currently existing GWA studies.Systems Biologynatural selection; adaptation; mutation; epistasis; fitness; polygenic
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Characterization and applications of novel DNA- and RNA-targeting CRISPR effectors
No full textThe development of molecular tools from natural discovery has played an integral role in the history of modern biology. Genome editing systems derived from the CRISPR bacterial immune system have enabled substantial increases in progress in applications such as forward and reverse genetics, protein tagging, gene therapy, and lineage tracing, among many others. While the initial DNA endonuclease family used in these contexts, Cas9, has demonstrated great utility, the discovery, characterization, and application of new programmable nucleic acid targeting systems could improve upon existing applications and provide new opportunities for the use of molecular tools. Here, we present several new families of single-effector CRISPR enzymes, including Cas12a and Cas13, and demonstrate their value for the targeting of either DNA or RNA in vivo and in vitro, for applications in genome editing, RNA knockdown, RNA editing, and diagnostics.
Chapter 1 provides an introduction to the CRISPR adaptive immune system, current approaches in genome editing, and a background of tools for RNA-targeting.
Chapter 2 describes the characterization of a novel single-effector CRISPR system, the type V system, and its associated nuclease, Cpf1/Cas12a. We demonstrate the targeting rules for Cpf1 in vitro and in vivo and apply it for genome editing.
Chapter 3 details the activity and targeting mechanism of the first RNA targeting CRISPR single-effector protein, C2c2/Cas13a, and reprograms it for targeting phage and transcripts in vivo. It also provides a biochemical characterization of Cas13a and describes the collateral effect, a promiscuous cleavage activity that is activated upon target binding in vitro.
Chapter 4 applies the collateral effect of Cas13a to generate a platform for sensitive and specific detection of nucleic acids, SHERLOCK. SHERLOCK is demonstrated for numerous diagnostic applications, including detection of viral and bacterial pathogens, SNP genotyping, and detection of mutations in circulating cell-free DNA.
Chapter 5 describes the engineering of the Cas13a system for in vivo RNA targeting in eukaryotic cells, and applies Cas13a as a tool for RNA knockdown, RNA binding, and RNA imaging.
Chapter 6 expands the in vivo applications of RNA targeting to the Cas13b and Cas13c families, and applies Cas13b for targeted editing of RNA transcripts in mammalian cells for potential therapeutic approaches.
Chapter 7 builds upon the SHERLOCK platform, characterizing the activities of a wide variety of Cas13a and Cas13b orthologs to provide in-sample multiplexing capabilities. It also describes the development of an instrument-free approach for detection based on lateral flow.
Chapter 8 provides an outlook on future developments in molecular tool development in DNA- and RNA-targeting systems and suggests new potential sources for molecular tools.Systems Biolog
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Clinical Sequencing Uncovers Origins and Evolution of Lassa Virus
Full text linkThe 2013-2015 West African epidemic of Ebola virus disease (EVD) reminds us how little is known about biosafety level-4 viruses. Like Ebola virus, Lassa virus (LASV) can cause hemorrhagic fever with high case fatality rates. We generated a genomic catalog of almost 200 LASV sequences from clinical and rodent reservoir samples. We show that whereas the 2013-2015 EVD epidemic is fueled by human-to-human transmissions, LASV infections mainly result from reservoir-to-human infections. We elucidated the spread of LASV across West Africa and show that this migration was accompanied by changes in LASV genome abundance, fatality rates, codon adaptation, and translational efficiency. By investigating intrahost evolution, we found that mutations accumulate in epitopes of viral surface proteins, suggesting selection for immune escape. This catalog will serve as a foundation for the development of vaccines and diagnostics.Organismic and Evolutionary BiologyAccepted Manuscrip
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A global reference for human genetic variation
Full text linkThe 1000 Genomes Project set out to provide a comprehensive description of common human genetic variation by applying whole-genome sequencing to a diverse set of individuals from multiple populations. Here we report completion of the project, having reconstructed the genomes of 2,504 individuals from 26 populations using a combination of low-coverage whole-genome sequencing, deep exome sequencing, and dense microarray genotyping. We characterized a broad spectrum of genetic variation, in total over 88 million variants (84.7 million single nucleotide polymorphisms (SNPs), 3.6 million short insertions/deletions (indels), and 60,000 structural variants), all phased onto high-quality haplotypes. This resource includes >99% of SNP variants with a frequency of >1% for a variety of ancestries. We describe the distribution of genetic variation across the global sample, and discuss the implications for common disease studies.Organismic and Evolutionary BiologyAccepted Manuscrip
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Genomic methods for the surveillance and epidemiologic inference of respiratory viral pathogens
No full textRespiratory viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV), are globally circulating pathogens with a substantial disease burden. Epidemiologic and genomic approaches to surveil respiratory viruses guide public health efforts and advance our understanding of viral evolution and transmission. However, interpreting respiratory viral surveillance data can be challenging, as data collection is often (i) biased, in that epidemiologic and genomic data are primarily derived from cases exhibiting moderate or severe disease; and (ii) dynamic, in that surveillance practices evolve in response to diagnostic practices, methodologic advances, and public health priorities. In this dissertation, I develop methods to mitigate the biases inherent in traditional respiratory viral surveillance approaches, and apply them to study the epidemiology and evolution of both SARS-CoV-2 and RSV. In Chapter 2, I use scalable genomic tools and samples from universal SARS-CoV-2 testing programs to conduct population-wide genomic surveillance, providing a comprehensive view of the Omicron variant’s arrival and spread in university communities. In Chapter 3, I develop an experimental protocol to sequence viral genomes from at-home rapid antigen tests, allowing genomic surveillance to extend to individuals who do not seek clinical diagnostic testing. In Chapters 4 and 5, I investigate the increase in RSV cases reported after the emergence of SARS-CoV-2. I demonstrate that increased respiratory viral testing, rather than the evolution of a more severe or transmissible RSV variant, can explain the apparent increase in cases. Moreover, I establish quantitative approaches to estimate epidemic parameters under the counterfactual scenario in which viral diagnostic testing remained unchanged. In summary, I develop robust genomic and epidemiologic surveillance strategies and demonstrate their ability to generate actionable insights into the evolution and transmission of respiratory viruses
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