The pitcher plant mosquito Wyeomyia smithii maintains a broad geographic range from the Gulf of Mexico to central Canada, and throughout its range is genetically and phenotypically variable, though fully interfertile. Many of the traits that vary across the broad range of this mosquito owe their diversity to selection on populations, which maximize fitness in the unique environment in which each populations finds itself. While a diversity of traits vary by latitude and merit the interest of evolutionary biologists, including critical photoperiod, voltinism, and thermal tolerance, of interest in the following thesis is the variation in blood-feeding propensity within this single species of mosquito. In no other mosquito species are some populations obligate non-biters while in other populations willingly hematophagous. This thesis explores the evolutionary transition from biting to non-biting in the pitcher plant mosquito at multiple levels of biological integration, starting first by establishing a heritable basis for the transition, then moving to the fitness and life historical consequences of both the natural system and of a line artificially selected in the lab. The latter half of this thesis moves on to probe the genetic architecture underlying the shift in phenotype and ends after examining the transition to non-biting at the level of the gene using an RNA-sequencing experiment. The results stemming from this thesis are thoroughly discussed: in short, we find that fitness differs between biting and non-biting populations, that complex genetic architectures underlie the transition to non-biting in nature, but not under artificial selection, and finally, that many candidate loci are differentially regulated in biting populations relative to non-biting populations and that these loci most often cluster with metabolic biological pathways.

Regulation of gene expression is an essential process for all living organisms. Transcriptional regulation, associated with chromatin, is governed by: (1) DNA sequence, which creates regulatory sites (promoters, enhancers and silencers), where sequence motifs and features (e. g. CpG) can attract transcription factors (TFs) and influence chromatin structure or RNA polymerase II (Pol II) binding, initiation and elongation; (2) non-sequence, epigenetic factors - histone modifications, TF binding, chromatin remodelling (histone placement, eviction and reconstitution), and non-coding RNA regulation. These factors interact with each other, creating a complex network of interactions. In this thesis I describe computational studies of heterochromatin factors in regulation of gene and repeat expression, an analysis of active regulatory elements, and global analyses of big datasets in C. elegans. I first show that a team of heterochromatin factors - HPL-2/HP1, LIN-13, LIN-61, LET-418/Mi-2, and H3K9me2 histone methyltransferase MET-2/SETDB1 - collaborates with piRNA and nuclear RNAi pathways to silence repetitive elements and protect the germline. I also found that the TACBGTA motif is particularly enriched on repeats and heterochromatin factors binding sites, and that repeat elements are derepressed in the soma during normal C. elegans ageing. I then describe the work on active regulatory regions. I show that CFP-1/CXXC1 binds CpG dense, nucleosome depleted promoters and, along SET-2, is required for H3K4me3 deposition at these loci. Using expression profiling I determined that the majority of CFP-1 binding targets are not significantly mis-regulated in cfp-1 mutants, but are weakly upregulated in bulk analyses. I also show that CFP-1 functionally interacts with the Sin3S/HDAC complex. In cfp-1 mutant I observed both loss and gain of SIN-3 binding, depending on chromatin context. Finally, I performed a data driven study on a large collection of ChIP-seq profiles using non-parametric sparse factor analyses (NSFA) and compared it to other, unsupervised machine learning algorithms. This study uncovered interactions and structure in genomic datasets. In addition, I present a collection of computational tools and methods I developed to facilitate processing, storage, retrieval, annotation, and analyses of large datasets in genomics.

Os microrganismos associados à cárie são, em sua maioria, microrganismos acidogênicos e acidúricos, anaeróbios estritos e facultativos. A presença de fungos é associada à microbiota de cárie radicular, sendo a espécie fúngica mais relacionada a Candida albicans. Embora estudos de cultivo e de análise de DNA comprovem a presença de fungos na microbiota associada a lesões de cárie radicular, demonstrando um gradiente crescente de colonização com a progressão da doença, pouco se sabe sobre o papel que estes microrganismos desempenham no processo de doença. O objetivo deste trabalho foi estudar o papel de Candida albicans na cárie radicular, através da análise de transcriptoma de biofilmes naturais de superfícies radiculares hígidas (n=10, SRS) e de lesão de cárie radicular ativa (n=9, RC). Foi avaliada a expressão diferencial de genes de Candida albicans, as funções específicas e vias metabólicas associadas a este microrganismo. O RNA total microbiano foi extraído e o mRNA isolado e sequenciado na plataforma Illumina Hi-Seq2500. Foram formados pool (grupamentos) das amostras com valores inferiores a 30ng/RNA para a construção de bibliotecas genômicas. Os dados gerados pelo sequenciamento de RNA-Seq foram compilados em uma tabela de contagem (reads) e mapeados com o genoma de referência (C. albicans SC5314) utilizando o software CLC Genomics Workbench 7.5.1. Para o cálculo do nível de expressão gênica os dados foram normalizados com o algoritmo DESeq. A expressão diferencial foi calculada com binomial negativa e False Discovery Rate (FDR<0,05). Os genes com maior expressão em RC e em SRS foram analisados pela mediana relativa de expressão (RME; Relative Median Expression) e expressão diferencial, assim como as vias metabólicas associadas a genes de virulência e metabolismo de açúcares. Dois genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) apresentaram expressão diferencial significativa em superfície radicular hígida (SRS) e tem suas funções relacionadas a formação de biofilme. Enquanto que em superfície radicular cariada (RC) sete genes (UTP20, FDR=0.018; ITR1, FDR=0.036; DHN6, FDR=0.046; CaO19.7197, FDR=0.046; CaO19.7838, FDR=0.046; STT4, FDR=0.046; GUT1, FDR=0.046) apresentaram expressão diferencial significativa e tem suas funções relacionadas a atividade metabólica, transporte de açúcares, tolerância ao estresse, invasão e regulação de pH. Candida albicans é um microrganismo importante no desenvolvimento da doença cárie radicular. / The microbiota associated with root caries must be acidogenic and aciduric. S. mutans, Lactobacillus, Actinomyces, Veilonella, Bifidobacterium, and other bacteria play important roles in root caries biofilm. Yeasts are also present on carious and non-carious root biofilms, being the specie Candida albicans the most prevalent yeast found in root biofilms. Although the presence of Candida albicans is stablished in the literature, and there are an increasing gradient of Candida species. colonization with caries progression, the role of this microorganism in root caries has not being totally elucidated. The aim of this study is to analyse the role of Candida albicans in root caries thought a transcriptomic analysis of biofilms of sound root surfaces (n=10, SRS) and root caries lesions (n=9, RC) using a high-throughput sequencing of cDNA (RNA-Seq). The differential expression of genes of Candida albicans SC5314, the specific functions and pathways associated with the gene expression of the present study were investigated. The total bacterial RNA was extracted and the mRNA was isolated (Illumina Hi-Seq2500). Samples with low RNA concentration (less than 30ng/RNA) were pooled, yielding a final sample size of SRS=10 and RC=9. Sequence reads were compiled in a count table and mapped to C. albicans SC5314 genome of reference, using the software CLC Genomics Workbench 7.5.1. Gene expression was calculated in the algorithm DESeq, and the differential expression was calculated with binomial negative (Log2FoldChange) and False Discovery Rate (FDR<0,05). The genes with higher expression in RC and SRS were analysed by the Relative Median Expression (RME), and the virulence factors and pathways and sugar metabolization related with Candida albicans pathogenicity in root caries were analysed. Two genes (CaO19.610, FDR=0.009; CaO19.2506, FDR=0.018) are up-regulated in Sound Root Surface (SRS) have their functions related to biofilm formation and seven (UTP20, FDR=0.018; ITR1, FDR=0.036; DHN6, FDR=0.046; CaO19.7197, FDR=0.046; CaO19.7838, FDR=0.046; STT4, FDR=0.046; GUT1, FDR=0.046) are up-regulated in biofilm of carious dentin (RC) have functions related to metabolic activity, sugar transport, stress tolerance, invasion and pH regulation.

Recent Next Generation Sequencing methods provide a count of RNA molecules in the form of short reads, yielding discrete, often highly non-normally distributed gene expression measurements. Due to this feature of RNA sequencing (RNA-seq) data, appropriate statistical inference methods are required. Although Negative Binomial (NB) regression has been generally accepted in the analysis of RNA-seq data, its appropriateness in the application to genetic studies has not been exhaustively evaluated. Additionally, adjusting for covariates that have an unknown relationship with expression of a gene has not been extensively evaluated in RNA-seq studies using the NB framework. Finally, the dependent structures in RNA-Seq data may violate the assumptions of some multiple testing correction methods. In this dissertation, we suggest an alternative regression method, evaluate the effect of covariates, and compare various multiple testing correction methods. We conduct simulation studies and apply these methods to a real data set. First, we suggest Firth’s logistic regression for detecting differentially expressed genes in RNA-seq data. We also recommend the data adaptive method that estimates a recalibrated distribution of test statistics. Firth’ logistic regression exhibits an appropriately controlled Type-I error rate using the data adaptive method and shows comparable power to NB regression in simulation studies. Next, we evaluate the effect of disease-associated covariates where the relationship between the covariate and gene expression is unknown. Although the power of NB and Firth’s logistic regression is decreased as disease-associated covariates are added in a model, Type-I error rates are well controlled in Firth’ logistic regression if the relationship between a covariate and disease is not strong. Finally, we compare multiple testing correction methods that control family-wise error rates and impose false discovery rates. The evaluation reveals that an understanding of study designs, RNA-seq data, and the consequences of applying specific regression and multiple testing correction methods are very important factors to control family-wise error rates or false discovery rates. We believe our statistical investigations will enrich gene expression studies and influence related statistical methods.

La régulation post-transcriptionnelle joue un rôle essentiel dans le système nerveux, de l’assemblage à la fonction. Chez les métazoaires, la famille multigénique ELAV / Hu code pour des protéines de liaison à l'ARN produites dans les neurones et impliqués dans cette régulation. Le gène elav (embryonic lethal abnormal visual system) code pour une protéine essentielle produite dans tous les neurones et est présent seulement chez les diptères. L’élucidation des fonctions des plus anciens paralogues de drosophile, fne (found in neurons) et rbp9 (RNA binding protein 9), a fait l'objet de ce travail, à cause de (1) l’étroite relation de ces gènes avec les orthologues de vertébrés, (2) la disponibilité de mutants nuls viables.En collaboration avec le laboratoire de Brian Oliver, NIH, États-Unis, nous avons utilisé le RNA-Seq pour analyser le transcriptome de tête de mutants fne-. Les données ont été analysées ) avec les programmes Cuffdiff et DESeq pour identifier des différences dans les niveaux de transcripts mutants et ceux d’une souche sauvage de référence (WT). L'épissage alternatif a été examiné avec Spanki (SPlicing ANalysis Kit), un suite de programmes que nous avons contribué à développer. Spanki utilise les données de séquence des jonctions exon-exon et compare l’abondance de formes alternatives et mutuellement exclusives de transcrits issus d’un événement d'épissage. Les premières analyses se sont concentrées sur les différences de transcriptomes entre males et femelles sauvages. La détermination sexuelle somatique et le comportement chez la drosophile sont sous le contrôle d'une cascade génétique bien caractérisée et contrôlée par les gènes Sxl, tra, msl2, dsx et fru, mais les cibles en aval de cette voie canonique demeurent largement inconnues. Notre approche nous a permis d’identifier (et de valider par qPCR) de nouveaux gènes exprimés différemment dans les deux sexes. J’ai montré que l’un d’entre eux est le gène fne, que sa régulation est indépendante de tra et tra2, mais dépend de Sxl. Ces propriétés font de fne la première cible identifiée dans une voie alternative de détermination sexuelle prédite par le laboratoire de T. Cline sur la base d'autres études. J'ai aussi montré l’existence d'un rôle précédemment non documenté pour tra et tra2 dans la lignée somatique mâle. Afin d'identifier des réseaux fonctionnels contrôlés par fne, les données du transcriptome de mutants ont été comparées à celles de la souche sauvage de référence. Plusieurs gènes dont le niveau de transcrits ou l’épissage alternatif est altéré en absence du gène fne ont été identifiés et validés par qPCR. Par exemple, l'épissage alternatif de unc-13, essentiel pour l'exocytose des vésicules, est affecté dans les mutants fne. D'autres gènes impliqués dans les fonctions synaptiques ont été identifiés, y compris n-syb, tomosyn, brp et Clc. En outre, il existe des interactions génétiques entre la mutation fne et des mutations affectant les fonctions synaptiques. Ces interactions peuvent causer une létalité synthétique dans les doubles mutants. Finalement, les males mutants fne- ont un comportement de « chaining» également compatible avec une fonction à la synapse. Ainsi, le gène fne établit un lien entre la régulation post-transcriptionnelle, la fonction synaptique et le comportement.Dans la dernière partie de mon travail, j'ai utilisé une approche évolutive pour tenter de distinguer les fonctions spécifiques de fne ou de rbp9, des fonctions ancestrales. Des approches moléculaires, anatomiques et comportementales ont été employées. Les résultats distinguent différentes catégories de gènes, spécifiquement ceux dont l'expression est affectée (1) uniquement par fne (2) uniquement par rbp9 (3) à la fois par fne et rbp9 de manière redondante ou synergique. J’ai mis en évidence une redondance fonctionnelle partielle des deux gènes causant une létalité synthétique à l'âge adulte dans les doubles mutants, mais j’ai aussi identifié des fonctions spécifiques. / Post-transcriptional regulation plays pivotal roles from assembly to function of the nervous system. In metazoans, the ELAV/Hu genes constitute a conserved multigene family of pan-neuronal RNA-binding protein involved in post-transcriptional regulation. The elav (embryonic lethal abnormal visual system) gene is the first described family member, it encodes a vital protein and is restricted to dipterans. Elucidating the functions of the more ancient Drosophila paralogs, fne (found in neurons) and rbp9 (RNA-binding-protein-9), has been the focus of this work, because of (1) the close relationship of these genes to the vertebrate orthologs, (2) the availability of viable null mutants.In collaboration with the laboratory of Brian Oliver, NIH, USA, we used RNA-Seq to analyze the head transcriptomes of fne- mutants and a wild type reference strain (WT). The RNA-Seq data was searched for differences in transcript levels using the programs Cuffdiff and DESeq. Alternative splicing was examined using a suite of programs called Spanki (SPlicing ANalysis Kit), whose development we participated in. Spanki utilizes only sequence reads spanning exon-junctions to compare pairs of mutually exclusive alternative splicing events.Initial analyses included sex-specific comparisons in WT transcriptomes. Somatic sexual determination and behavior in Drosophila are under the control of a well characterized genetic cascade initiated by Sxl (Sex lethal), but the targets downstream of this canonical pathway remain largely unknown. As expected, the genes Sxl, tra (transformer), msl2 (male-specific lethal gene), dsx (doublesex) and fru (fruitless), which belong to the canonical sex-determination/dosage compensation pathways were identified by our analyses. In addition, new genes whose transcript expression levels differ between the sexes were identified and validated by qPCR. Further, I obtained evidence for previously undocumented roles of tra and tra2 (transformer 2) in the male soma. Finally, a sex-biased alternative splicing event was identified in fne, whose regulation is independent of tra or tra2, but dependent upon Sxl. This makes of fne the first Sxl-dependent, tra/tra2-independent target identified in a sex determination/differentiation pathway, previously been predicted to exist based upon other studies.The data was analyzed to identify functional networks controlled by fne. I found that it affects the expression of several genes at the level of transcript expression and alternative splicing involved in synaptic functions. They include of unc-13, n-syb (neuronal Synaptobrevin), tomosyn, brp (bruchpilot) and Clc (Clathrin light chain). Further, genetic interactions between fne and shi (shibire) or nrg (neuroglian), which also possess synaptic functions, reveal synthetic lethality in the double mutants. In addition, fne mutant males display a fruitless-like chaining behavior, also consistent with a function at the synapse. Thus the fne gene links post-transcriptional regulation to synaptic function and behavior.Finally, I used an evolutionary approach to distinguish newly evolved functions, i.e. specific to fne or rbp9, from those that are shared and thus more likely to be ancestral. Molecular, anatomical and behavioral approaches in parallel analyses of fne and rbp9, show that they possess both gene-specific and overlapping functions. The latter is evident from synthetic lethality in early adulthood of double mutants. My results distinguish different gene categories with respect to their regulation: genes whose expression is affected (1) only by fne (2) only by rbp9 (3) both by fne and rbp9 in redundant or synergistic ways. Finally, I showed that male-male interactions dramatically differ between fne versus rbp9 mutants, revealing the emergence of a new (or loss of an ancestral) function in behavior.

Eukaryotes have developed stringent regulatory mechanisms that control cell division and ensure proper chromosome segregation. Maintaining genome integrity is especially important during meiosis, the specialised cell division programme in the germline that generates haploid gametes. As these cells transmit genetic information to the next generation, the consequences of meiotic errors are not restricted to an organismal level, but can directly impact the fitness of the offspring. Mammals display a high degree of sexual dimorphism in meiosis with regard to the stringency of regulatory mechanisms. This manifests in a relatively high degree of maternally-derived aneuploidies due to weaker checkpoint control in females, whereas more rigorous checkpoints in males frequently perturb fertility. Mouse models of aneuploidy often exhibit complete male sterility and early germ cell arrest, preventing the study of aneuploidy during late and post-meiotic stages in males. In this thesis, we have used the trans-chromosomic mouse model, Tc1, which carries a single copy of human chromosome 21 (HsChr21) and show that, unlike other aneuploid mouse strains, the Tc1 mouse can successfully passage the exogenous human chromosome through male meiosis and generate aneuploid offspring. Our investigations have shown that the presence of the aneuploid human chromosome causes spermatogenic defects due to an arrest at the first meiotic division. Despite this impairment, we found an unexpectedly high number of aneuploid gametes in Tc1 males and the majority of males were able to produce aneuploid offspring, albeit at a lower frequency. Transmission of HsChr21 through the male germline was less efficient compared to female germline transmission, but allowed us to study the impact of male germline-associated chromatin remodelling on the transcriptional deployment of HsChr21 in the offspring. This revealed that, despite fundamentally different developmental dynamics, male- versus female-germline passage result in indistinguishable transcriptional and regulatory phenotypes. An important pathway in the male germline involves the expression of piRNAs, a class of small non-coding RNAs that are commonly found in the germline of animals where they defend cells against transposable elements. Profiling the expression of small RNAs in the Tc1 mouse showed that conserved human piRNA clusters can be successfully transcribed by the mouse piRNA machinery. In addition, we detected Tc1-specific piRNA sequences that were neither present in human nor mouse, mapping to a human-specific repeat element. In line with the previously observed activation of human-specific repeat elements in the Tc1 mouse, this suggests that novel transcripts arising from human repeats can trigger an adaptive piRNA response, thereby demonstrating the plasticity of this pathway to newly invading repeat elements. Transcriptional profiling of spermatogenic cell populations on a single-cell level allowed us to generate an atlas of gene expression over the course of spermatogenesis and dissect meiotic silencing dynamics in the presence of aneuploidy. Transcriptional silencing during meiosis occurs in response to unpaired chromosomes and, in male germ cells, affects the sex chromosomes due to their largely unpaired nature. We found that the presence of HsChr21 has no impact on the silencing of chromosome X, however, the two chromosomes display drastically different silencing patterns with HsChr21 showing a much weaker repression. Taken together, this study revealed a higher than expected tolerance for aneuploidy in the mouse male germline thus allowing the characterisation of meiotic checkpoint mechanisms, the meiotic silencing response to unpaired chromosomes as well as piRNA expression in the presence of an exogenous human chromosome.

Orientador: Alexandrina Sartori / Resumo: A leishmaniose visceral (LV) é uma doença negligenciada causada por Leishmania donovani na Índia e África ou Leishmania infantum na Europa e América Latina. O desenvolvimento de resposta imune eficaz e subsequente eliminação destes patógenos, requer o reconhecimento inicial da Leishmania, o qual é intermediado por receptores de reconhecimento padrão expressos por células da imunidade inata, entre eles os receptores de ligação a nucleotídeo (NLRs). Alguns NLRs ativam uma plataforma de proteínas, os inflamassomas, responsáveis pela ativação da caspase-1 e maturação de IL-1β e IL-18 e outra classe de NLRs, chamada NODs, ativam vias que culminam na ativação de NF-κB e produção de mediadores inflamatórios. O envolvimento desses receptores na LV ainda é pouco elucidado. Mesmo diante dos mecanismos de defesa do hospedeiro, esses parasitas conseguem sobreviver dentro dos macrófagos utilizando várias estratégias para escapar da resposta imune. Para um melhor entendimento dos mecanismos imunes envolvidos na LV, caracterizamos o perfil transcricional e a formação de inflamassomas e NODsomas de células THP-1 infectadas com L. infantum. Os resultados mostram que a L. infantum não induziu produção de TNF-α, IL-6 e IL-1β e nem ativação de caspase-1 após 8, 24 e 48 horas de infecção. Além disso, a infecção resultou em padrão de expressão gênica similar às células sem estímulo e distinto de células estimuladas com LPS, indicando que os parasitas entram nas células de forma mais silenciosa. Ap... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Visceral leishmaniasis (VL) is a neglected infectious disease caused by Leishmania donovani in India and Africa or Leishmania infantum in Europe and Latin America. The development of an effective immune response and subsequent elimination of these pathogens requires the initial recognition of the Leishmania that is mediated by pattern recognition receptors expressed in innate immunity cells, such as nucleotide-binding receptors (NLRs). Some NLRs activate a multiprotein platform named inflammasomes, responsible for the activation of caspase-1 and consequent maturation of IL-1β and IL-18; and another class of NLRs, the NODs, activate pathways that trigger NF-κB activation and production of inflammatory mediators. The involvement of NLRs in LV is poorly elucidated. Even in the presence of host defense mechanisms, these parasites can survive within the macrophages by employing successful strategies to escape from immune response. For a better understanding of the immune mechanisms involved in LV, we characterized the transcriptomic profiling and assembly of inflammasomes and NODsomas during infection with L. infantum in THP-1 cells. The results show that L. infantum did not induce the production of TNF-α, IL-6 and IL-1β nor activation of caspase-1 after 8, 24 and 48 hours of infection. In addition, the infection resulted in a pattern of gene expression similar to the non-stimulated cells and distinct from LPS-stimulated cells, indicating that the parasites enter inside cells in a... (Complete abstract click electronic access below) / Doutor

The aim of this project was to investigate the transcriptome of human haematopoietic stem cells (HSCs), megakaryocytes and platelets to gain insights into steady state and accelerated thrombopoiesis that occurs in states of haemostatic demand and in thrombosis by applying these findings to the pathological setting of acute coronary thrombosis. To investigate transcriptional heterogeneity within the human HSC population, single cell RNA sequencing was performed in human bone marrow HSCs. Transcriptionally distinct subpopulations were identified including two megakaryocyte biased subsets with potentially differing functional relevance. Both populations expressed megakaryocyte specific transcripts, one of which also co-expressed common myeloid and megakaryocyte-erythroid progenitor transcripts while the other did not. This study represents the first interrogation of the human bone marrow megakaryocyte transcriptome. Cells were collected from healthy human bone marrow and analysed by low input and single cell RNA sequencing. To identify novel drivers of megakaryocyte maturation, the human bone marrow megakaryocyte transcriptome was compared to that of megakaryocytes cultured from human CD34+ cells, a process known to generate immature megakaryocytes. Transcriptional signatures associated with increasing megakaryocyte ploidy were then investigated. Increasing megakaryocyte ploidy level was found to be associated with an upregulation of transcripts involved in translation and protein processing as well as expression of a number of transmembrane receptors which might have functional relevance. Finally, the pathological setting of acute coronary thrombosis was used as a model for accelerated thrombopoiesis. Megakaryocyte and platelet transcriptomes were compared between patients with acute myocardial infarction (AMI) as well as severe coronary disease and a control group. The transcriptional signature relating to disease compared to control in megakaryocytes included upregulation of platelet activation related transcripts in megakaryocytes isolated from patients with AMI and severe coronary artery disease.

During the Human Genome Project the first hundred billion bases were sequenced in four years, however, the second hundred billion bases were sequenced in four months (NHGRI, 2013). As efforts were made to improve every aspect of sequencing in this project, cost became inversely proportional to the speed (NHGRI, 2013). Human Genome Project ended in April 2003 but research in faster and cheaper ways to sequence the DNA is active to date (NHGRI, 2013). On the one hand, these advancements have allowed the convenient and unbiased generation and interrogation of a variety of omics datasets; on the other hand, they have substantially contributed towards the ever-increasing size of biological data. Therefore, informatics techniques are indispensable tools in the field of biology and medicine due to their ability to efficiently store and probe large datasets. Bioinformatics is a specialized domain under informatics that focusses on biological data storage, organization and analysis (NHGRI, 2013). Here, I have applied informatics approaches such as database designing and web development in the context of biological datasets or bioinformatics, to create a novel web-based resource that allows users to explore the comprehensive transcriptome of common aquatic tunicate named Oikopleura dioica (O .dioica), and access their associated annotations across key developmental time points, conveniently. This unique resource will substantially contribute towards studies on development, evolution and genetics of chordates using O. dioica as a model. Mendelian or single-gene disorders such as cystic fibrosis, sickle-cell anemia, Huntington’s disease, and Rett’s syndrome run across generations in families (Chial, 2008). Allelic variations associated with Mendelian disorders primarily reside in the protein-coding regions of the genome, collectively called an exome (Stenson et al., 2009). Therefore, sequencing of exome rather than whole genome is an efficient and practical approach to discover etiologic variants in our genome (Bamshad et al., 2011). Renal agenesis (RA) is a severe form of congenital anomalies of the kidney and urinary tract (CAKUT) where children are born with one (unilateral renal agenesis) or no kidneys (bilateral renal agenesis) (Brophy et al., 2017; Yalavarthy & Parikh, 2003). In this study, we have applied exome-sequencing technique to selective human patients in a renal agenesis (RA) pedigree that followed a Mendelian mode of disease transmission. Exome sequencing and molecular techniques combined with my bioinformatics analysis has led to the discovery of a novel RA gene called GREB1L (Brophy et al., 2017). In this study, we have successfully demonstrated the validation of exome sequencing and bioinformatics techniques to narrow down disease-associated mutations in human genome. Additionally, the results from this study has substantially contributed towards understanding the molecular basis of CAKUT. Discovery of novel etiologic variants will enhance our understanding of human diseases and development. High-throughput sequencing technique called RNA-Seq has revolutionized the field of transcriptome analysis (Z. Wang, Gerstein, & Snyder, 2009). Concisely, a library of cDNA is prepared from a RNA sample using an enzyme called reverse transcriptase (Nottingham et al., 2016). Next, the cDNA is fragmented, sequenced using a sequencing platform of choice and mapped to a reference genome, assembled transcriptome, or assembled de novo to generate a transcriptome (Grabherr et al., 2011; Nottingham et al., 2016). Mapping allows detection of high-resolution transcript boundaries, quantification of transcript expression and identification of novel transcripts in the genome. We have applied RNA-Seq to analyze the gene expression patterns in water flea otherwise known as D. pulex to work out the genetic details underlying heavy metal induced stress (unpublished) and predator induced phenotypic plasticity (PIPP) (Rozenberg et al., 2015), independently. My bioinformatics analysis of the RNA-Seq data has facilitated the discovery of key biological processes participating in metal induced stress response and predator induced defense mechanisms in D. pulex. These studies are great additions to the field of ecotoxicogenomics, phenotypic plasticity and have aided us in gaining mechanistic insight into the impact of toxicant and predator exposure on D. pulex at a bimolecular level.

Over the past decade, the evolution of next-generation sequencing technology has considerably advanced the genomics research. As a consequence, fast and accurate computational methods are needed for analyzing the large data in different applications. The research presented in this dissertation focuses on three areas: RNA-seq read mapping, large-scale data query, and metagenomics sequence classification. A critical step of RNA-seq data analysis is to map the RNA-seq reads onto a reference genome. This dissertation presents a novel splice alignment tool, MapSplice3. It achieves high read alignment and base mapping yields and is able to detect splice junctions, gene fusions, and circular RNAs comprehensively at the same time. Based on MapSplice3, we further extend a novel lightweight approach called iMapSplice that enables personalized mRNA transcriptional profiling. As huge amount of RNA-seq has been shared through public datasets, it provides invaluable resources for researchers to test hypotheses by reusing existing datasets. To meet the needs of efficiently querying large-scale sequencing data, a novel method, called SeqOthello, has been developed. It is able to efficiently query sequence k-mers against large-scale datasets and finally determines the existence of the given sequence. Metagenomics studies often generate tens of millions of reads to capture the presence of microbial organisms. Thus efficient and accurate algorithms are in high demand. In this dissertation, we introduce MetaOthello, a probabilistic hashing classifier for metagenomic sequences. It supports efficient query of a taxon using its k-mer signatures.