Genome annotation for clinical genomic diagnostics: strengths and weaknesses

The Human Genome Project and advances in DNA sequencing technologies have revolutionized the identification of genetic disorders through the use of clinical exome sequencing. However, in a considerable number of patients, the genetic basis remains unclear. As clinicians begin to consider whole-genome sequencing, an understanding of the processes and tools involved and the factors to consider in the annotation of the structure and function of genomic elements that might influence variant identification is crucial. Here, we discuss and illustrate the strengths and weaknesses of approaches for the annotation and classification of important elements of protein-coding genes, other genomic elements such as pseudogenes and the non-coding genome, comparative-genomic approaches for inferring gene function, and new technologies for aiding genome annotation, as a practical guide for clinicians when considering pathogenic sequence variation. Complete and accurate annotation of structure and function of genome features has the potential to reduce both false-negative (from missing annotation) and false-positive (from incorrect annotation) errors in causal variant identification in exome and genome sequences. Re-analysis of unsolved cases will be necessary as newer technology improves genome annotation, potentially improving the rate of diagnosis

The Genome Sequence of the Grape Phylloxera Provides Insights into the Evolution, Adaptation, and Invasion Routes of an Iconic Pest

Background: Although native to North America, the invasion of the aphid-like grape phylloxera Daktulosphaira vitifoliae across the globe altered the course of grape cultivation. For the past 150 years, viticulture relied on grafting-resistant North American Vitis species as rootstocks, thereby limiting genetic stocks tolerant to other stressors such as pathogens and climate change. Limited understanding of the insect genetics resulted in successive outbreaks across the globe when rootstocks failed. Here we report the 294-Mb genome of D. vitifoliae as a basic tool to understand host plant manipulation, nutritional endosymbiosis, and enhance global viticulture. Results: Using a combination of genome, RNA, and population resequencing, we found grape phylloxera showed high duplication rates since its common ancestor with aphids, but similarity in most metabolic genes, despite lacking obligate nutritional symbioses and feeding from parenchyma. Similarly, no enrichment occurred in development genes in relation to viviparity. However, phylloxera evolved > 2700 unique genes that resemble putative effectors and are active during feeding. Population sequencing revealed the global invasion began from the upper Mississippi River in North America, spread to Europe and from there to the rest of the world. Conclusions: The grape phylloxera genome reveals genetic architecture relative to the evolution of nutritional endosymbiosis, viviparity, and herbivory. The extraordinary expansion in effector genes also suggests novel adaptations to plant feeding and how insects induce complex plant phenotypes, for instance galls. Finally, our understanding of the origin of this invasive species and its genome provide genetics resources to alleviate rootstock bottlenecks restricting the advancement of viticulture

Microbial Genome Evolution Due to Multifaceted Symbiosis within the Tsetse Fly (Diptera: Glossinidae)

Microbes are capable of rapid genetic modification, enabling the habitation of a wide field of niches, including forming interdependent associations with macroscopic hosts. While ancient multipartite mutualisms have been shown to involve metabolic complementation, little is known concerning the early genomic adaptations leading towards co-residence within a novel host. The overall objective of this research is to gain insight on genome evolution resulting from symbiosis, particularly by examining bacteria with varying levels of host dependency and times of establishment. The tsetse fly (Diptera: Glossinidae) serves as a relatively simple model system to investigate evolutionary aspects of symbiosis, while also maintaining medical and agricultural significance as vectors of African trypanosomes. In addition to potentially harboring trypanosomes, the tsetse enteric microbiota consists of two gamma-Proteobacteria: the anciently associated obligate mutualist Wigglesworthia spp. and the recently established commensal Sodalis glossinidius. The genomes of Wigglesworthia spp. (isolated from Glossina morsitans (Wgm) and G. brevipalpis (Wgb)), Sodalis and Trypanosoma brucei subspp. have been sequenced and annotated, facilitating empirical studies exploring potential partner interactions and adaptations. My work first examines the importance of nutrient provisioning, specifically thiamine (Vitamin B1), for the maintenance of a stable symbiotic environment within the tsetse host. These studies demonstrated that Sodalis required exogenous thiamine for proliferation due to the erosion of biosynthetic capabilities, while Wigglesworthia thiamine biosynthetic loci expression was influenced by the functional demand for this nutrient. My research also explored how distinct symbiont metabolic capabilities, retained by Wgm, but lacking in the Wgb genome, contribute to host biology and phenotypic variation. Wgm chorismate and folate (Vitamin B9) biosynthesis increased during times of nutrient stress, such as pregnancy and trypanosome infection, and was found to be critical for host biology. Lastly, genetic adaptations leading towards symbiont diversification and establishment in novel hosts were investigated. To accomplish this, molecular phylogenetic analyses were performed on Sodalis and closely related bacteria using genome regions traditionally associated with accelerated evolution, such as surface encoding loci and internal transcribed spacer regions, further increasing the resolution of this clade. This enhanced knowledge of tsetse symbionts increases our understanding of tsetse biology, potentially contributing to disease control strategies, and offers additional insights regarding fundamental evolutionary aspects involved in microbial symbiosis

Exploring the boundaries of shallow phylogeny in the YESS group and the dynamics of gene cluster and operon formation in bacterial genomes

In this thesis I look at two different problems in bacterial genomic analysis. The first involves reconstructing the evolutionary history between a group of closely related bacteria. I addressed whether or not it is possible to separate such genomes into different genera, species and strains. Specifically, I addressed how different approaches such as the use of 16S rRNA phylogenetic trees, phylogenetic supertrees and concatenation of individual genes in order to construct phylogenetic trees compare with one another. What effect will problems associated with resolving shallow-phylogeny have on recovering a tree of life? Ultimately I show that for the group of genomes involved, different methods and data produce different results and that the true tree, if a tree-like structure does indeed exist for these genomes, is unrecoverable using such approaches. In the second part of my thesis I examine the phenomenon of gene clustering in bacterial genomes. I present a software program, GenClust, for the identification, analysis and visualisation of gene clusters. I show how GenClust can be used to recover and analyse clusters of genes involved in amino acid biosynthesis across a large !-proteobacterial dataset. Finally, I examine models of gene cluster and operon formation and test them with real data, using a combined approach of comparing clusters on both structural similarity and the underlying phylogenetic signals of the clustered genes. I provide a hypothesis for the selective forces driving cluster and operon formation in bacterial genomes

The evolution of immune genes in tsetse flies (Glossina) and insights into tsetse-symbiont-trypanosome interactions

Tsetse flies (genera Glossina) are the sole biological vectors of African Trypanosoma species, the infectious agents of African Trypanosomiasis. Vector control is a key inhibitor of disease transmission; however, long-term control measures are economically and ecologically unsustainable and therefore, alternatives must be explored. In this thesis we aim to explore the evolution of three important immune genes: attacin-A (AttA), Defensin (Def) and Toll-like receptor 2 (TLR2), in relation to symbionts and parasitic interactions. This could in turn lay the foundations for genetic control methods The successful identification of novel attacin orthologues confirmed the previous descriptions of attacin clusters within the Glossina genome, while a single novel defensin orthologue was identified in each of the six Glossina genomes. A total of six TLRs were confirmed within the Glossina genome, and three additional TLRs were potentially identified, though these are unconfirmed. The evolutionary history of the attacin cluster remains undetermined, however concerted evolution likely impacts the evolution of AttA, while Def and TLRs are governed by strict Darwinian selection. A wild population sample of Glossina morsitans morsitans illustrated differing levels of nucleotide variation in each gene, Def being the least polymorphic (n = 8) and TLR2 being the most (n = 22). All genes indicated a recent population expansion event and deviations from neutrality, indicative of population expansion and balancing selection. Genetic variation in both AttA and TLR2 was found to be maintained via purifying selection, while Def exhibited signs of the Red Queen arms race and balancing section. Trypanosome infection rates were unexpectedly high (69.35%), consisting of mixed species infections. Advantageous Def variants were observed to reduce infection rates within samples, while an observable relationship between TLR2 and symbiont variation, and infection rate requires further research. The results within described the impacts of evolution and population change on immune genes and how the interactions with symbiont populations can influence trypanosome infection rates. This thesis indicates that an understanding of the evolution and interactions of the tsetse-symbiont-trypanosome triplet could be used to inform novel genetic control methods

Characterization of host-symbiont molecular interactions and evolutionary relationships in the gutless oligochaete Olavius algarvensis

The marine gutless oligochaete O. algarvensis lives in obligate symbiosis with a chemosynthetic bacterial consortium that exclusively provides it with nutrition. This thesis contributes to a better understanding of how this essential symbiosis is maintained, both on a physiological and immunological level (chapter IV), as well as from an evolutionary perspective (chapter II). This is addressed by using metagenomics, -proteomics and -transcriptomics to better understand symbiont transmission, diversity, co-divergent evolution and the molecular adaptations of the host that allow it to intimately associate with a diverse and physiologically demanding chemosynthetic consortium (anoxia and noxious substances). Furthermore, chapter III provides the first functional genomic description of the spirochaetal symbiont of O. algarvensis, showing that it is most likely a beneficial symbiont involved in the utilization and funneling of environmentally derived organic nutrients into the symbiosis

Genomic diversity in naturally transformable Streptococcus pneumoniae

Infections due to Streptococcus pneumoniae (the pneumococcus) remain a substantial source of morbidity and mortality in both developing and developed countries despite a century of research and the development of effective therapeutic interventions (such as antibiotic therapy and vaccination). The ability of the pneumococcus to evade multiple classes of antibiotic through several genetically determined resistance mechanisms and its evasion of capsular polysaccharide based vaccines through serotype replacement and capsular switching, all reflect the extensive diversity and plasticity of the genome of this naturally transformable organism which can readily alter its genome in response to its environment and the pressures placed upon it in order to survive. The purpose of this thesis is to investigate this diversity from a genome sequence perspective and to relate these observations to pneumococcal molecular epidemiology in a region of high biodiversity, the pathogenesis of certain disease manifestations and assess for a possible bacterial genetic basis for the pneumococcal phenotypes of, “carriage” and, “invasion.” In order to do this, microarray comparative genomic hybridization (CGH) has been utilized to compare DNA from a variety of pneumococcal isolates chosen from 10 diverse serotypes and Multilocus Sequence Types and from clinically relevant serotypes and sequence types (particularly serotypes 3, 4 and 14 and sequence types ST9, ST246 and ST180)) against a reference, sequenced pneumococcal genome from an extensively investigated serotype 4 isolate – TIGR4. Microarray comparison of the transcriptional profiles of several isolates has also been undertaken to compare gene expression from isolates of serotype 1 (ST227 and ST306) and serotype 3 (ST180) related to particular disease states and exposure of a multi-resistant pneumococcus to an antimicrobial (clarithromycin) commonly used to treat pneumococcal pneumonia

Genetic Studies of Wildlife

Genetic techniques are being more frequently used to understand the biology and management of wildlife species. The wild turkey is one species of genetic interest because the correct identification of individuals to the subspecies level is difficult using traditional methods. Currently phenotypic differences in plumage, especially the upper tail coverts, are used to assign individuals to subspecies. To hunters wanting to complete a “grand slam,” identification of birds’ subspecies is important. This study focuses on the five extant subspecies: Eastern (M. g. silvestris), Osceola (M. g. osceola), Rio Grande (M. g. intermedia), Merriam’s (M. g. merriami), and Gould’s (M. g. mexicana). I aimed to determine if molecular genetic data provide support for currently recognized subspecies. I also attempted to determine if quantitative measurements of coloration of the upper tail coverts is geographically discrete and consistent with historical subspecies boundaries. I used primer sets for 11 single nucleotide polymorphisms thought to be diagnostic at the subspecies level and sequenced DNA of tissue samples from 81 birds to determine whether they were pure examples of a subspecies or hybrids. To measure plumage coloration, I used a spectrophotometer to obtain quantitative measurements of upper tail coverts from individuals obtained in 21 states and all subspecies. Genetic analyses suggested that most wild turkeys in Nebraska represent a mixture of many subspecies. Morphological analyses indicated that there are not five distinct spectral ranges that correspond with accepted subspecies, but most likely two that roughly divide turkeys from east to west. These analyses plus comparison of mitochondrial genomes suggests that the genetic landscape of wild turkey is basically divided into eastern and western groups. To explore the use of molecular phylogenetics in wildlife genetics I also did a study on the evolution of Transmissible Spongiform Encephalopathies across 102 species of mammals. Phylogenetic hypotheses for the prion protein gene, thought to be responsible for transmissible spongiform encephalopathies, and a species tree inferred from 20 unlinked nuclear genes, were compared, finding highly congruent topologies. Mapping the presence/absence of TSEs on the species tree, TSEs occur non-randomly and have arisen independently and recently in different mammalian groups. This suggests that the evolution of TSEs develops in groups of species irrespective of PRNP genotype. Advisor: Robert M. Zin