The human genome consists of over 3 billion nucleotides and would be around 2 meters long if uncoiled and laid out. Each human somatic cell contains all this in their nucleus which is only around 5 µm across. This extreme compaction is largely achieved by wrapping the DNA around a histone octamer, the nucleosome. Still, the DNA is accessible to the transcriptional machinery and this regulation is highly dynamic and change rapidly with, e.g. exposure to drugs. The individual histone proteins can carry specific modifications such as methylations and acetylations. These modifications are a major part of the epigenetic status of the DNA which contributes significantly to the transcriptional status of a gene - certain modifications repress transcription and others are necessary for transcription to occur. Specific histone methylations and acetylations have also been implicated in more detailed regulation such as inclusion/exclusion of individual exons, i.e. splicing. Thus, the nucleosome is involved in chromatin remodeling and transcriptional regulation – both directly from steric hindrance but also as a signaling platform via the epigenetic modifications. In this work, we have developed tools for storage (Paper I) and normalization (Paper II) of next generation sequencing data in general, and analyzed nucleosome locations and histone modification in particular (Paper I, III and IV). The computational tools developed allowed us as one of the first groups to discover well positioned nucleosomes over internal exons in such wide spread organisms as worm, mouse and human. We have also provided biological insight into how the epigenetic histone modifications can control exon expression in a combinatorial way. This was achieved by applying a Monte Carlo feature selection system in combination with rule based modeling of exon expression. The constructed model was validated on data generated in three additional cell types suggesting a general mechanism.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several tumours and hyperproliferative disorders. Hypoxia plays an important role in KSHV lifecycle, as hypoxia-inducible factors (HIFs) are involved in the latent/lytic switch and affect other KSHV genes, and as KSHV infection can in turn enhance cellular levels of HIFs. Two KSHV-associated tumours tend to develop in settings of relative hypoxia; Kaposi's sarcoma (KS) often occurs in the lower extremities and primary effusion lymphoma (PEL) exists in pleural effusions. A better knowledge of the pathways that regulate KSHV infection in hypoxia is therefore essential for an improved understanding of viral infection and pathogenesis. MicroRNAs (miRNAs) have been shown to play important roles in regulating the expression of genes in oncogenesis, and herpesviruses, including KSHV, encode for miRNAs. This thesis describes a multidisciplinary approach toward understanding the mechanisms behind the hypoxia-regulated miRNA-mRNA networks in the context of KSHV infection. The question of miRNA and mRNA regulation through hypoxia, KSHV or both is addressed in this thesis by deep sequencing and gene expression assays as well as various transfection and functional assays. In chronically infected cells compared to uninfected controls, it is demonstrated that the majority of cellular miRNAs whose expression is affected are substantially down-regulated. A third of this down-regulation can be attributed to a single genomic region, 14q32 cluster, where miRNAs are lowly expressed in infected cells. In hypoxia, hsa-miR-210 is the only miRNA to be consistently up-regulated in the KSHVinfected cell lines subjected to deep sequencing in this study. Computational approaches additionally allowed for the investigation of mRNA targets. Inversely correlated miRNAmRNA target pairs were identified and distributed into canonical pathways and biological networks. Taken together, these results suggest that miRNAs affected by hypoxic stress and/or viral infection are implicated in the pathogenesis of KSHV-related diseases. It is expected that the outcomes of these studies will change our understanding of how KSHV uses the host RNA silencing machinery to its advantage and how this intersects with the use of the cell's response to hypoxia.

This diploma thesis deals with a development of a strategy for data evaluation generated by next-generation sequencing. Using bioinformatics tools such as Galaxy, Velvet and Enterovirus genotyping tool new aproach of data processing was optimized. There were 22 samples analyzed which of 10 were grown on cell culture. Remaining 12 were obtained from real stool samples. All samples were taken from children at the highest genetic risk of type 1 diabetes. All of them were enterovirus positive. Enteroviruses and their following infections have been suspecting to be involved in ehiology of type 1 diabetes for a long time. That's a disease resulting to an absolut insulin deficiency due to autoimmune destruction of pancreatic beta cells. Genetic components seems to be relatively well defined (the HLA, INS, STLA4, PTPN22, CTLA4, IFIH1 and numerous other genes), the environmental part of the etiology remains obscured. We were able to assemble 22 genomes de novo. However, there were numerous gaps among the particular contigs. For the first nine samples these gaps were complemented by Sanger sequencing. Nine full-length genomes were assempled this way. The main contribution of this work was to create a universal process of analyzing data from next-generation sequencing. This has already been using for further...

Recently, examination of deoxyribonucleic acids in water samples (environmental DNA or eDNA) has shown promise for identifying fish species present in water bodies. In water, eDNA arises from bodily secretions such as mucus, gametes, and feces. I investigated whether eDNA can be effective for characterizing fish presence, relative abundance, biomass, and species composition in a large Arizona reservoir (Theodore Roosevelt Lake) and 12 small Arizona (<24 ha) waterbodies. Specifically, I compared fish presence, relative abundance (catch per unit effort [CPUE]), biomass (biomass per unit effort [BPUE]), and species composition measured through eDNA methods and established American Fisheries Society (AFS) standard sampling methods in Theodore Roosevelt Lake and 12 small waterbodies. Environmental DNA sampling resulted in detection of Gizzard Shad Dorosoma cepedianum at a higher percentage of sites than boat electrofishing, both in spring and fall. Contrarily, gill nets detected Gizzard Shad at more sites than eDNA for both spring and fall sampling in Lake Roosevelt. Boat electrofishing and gill netting detected Largemouth Bass Micropterus salmoides at more sites than eDNA, with the exception of fall gill net sites which equally detected Largemouth Bass at sites within Lake Roosevelt. Environmental DNA detected Largemouth Bass and Bluegill Lepomis macrochirus at more Arizona small lakes than detection with established gear methods. I observed no relationship between relative abundance and biomass of Largemouth Bass and Gizzard Shad measured by established methods and their DNA copies at individual sites or by lake section in Lake Roosevelt. Likewise, I found no relationship between relative abundance and biomass of Largemouth Bass and Bluegill measured by established methods and their DNA copies across 12 small waterbodies. Plot analysis conceivably illustrated that reservoir-wide catch composition (numbers and total weight of fish [g]) achieved through a combination of gear types (boat electrofishing + gill netting) for Largemouth Bass and Gizzard Shad was slightly similar to the proportion of total eDNA copies of each species for both spring and fall field sampling. Likewise, spring and fall gill net surveys somewhat portrayed total catch composition (numbers and total weight of fish [g]) of Largemouth Bass and Gizzard Shad similar to the proportion of total eDNA copies of each species. The exception was the total lack of similarity illustrated between proportions of fish caught in spring and fall boat electrofishing and total eDNA copies of each species in Lake Roosevelt. However, the deceptive similarity of all the plots were not present in the chi-square analysis with the exception of fall gill net surveys in Lake Roosevelt. In addition, eDNA did reflect the relative proportions of Largemouth Bass and Bluegill in total catch composition in some, but not all of 12 small Arizona waterbodies. The ease of eDNA sampling over established fish sampling makes it appealing to natural resource managers. Compared to current established fish sampling methods, eDNA sampling can be less laborious, less time consuming, and more cost effective. Environmental DNA sampling may be useful in sites that have difficult access such as remote sites. However, evaluation of eDNA is necessary to identify limitations and benefits in fish monitoring programs. Furthermore, field sampling protocols, filtration, DNA extraction, primer design, and DNA sequencing methods need further refinement and testing before incorporation into standard fish sampling surveys.

The main theme of this dissertation is to develop a distributed computational pipeline for processing next-generation RNA sequencing (RNA-seq) data. RNA-seq experiments generate hundreds of millions of short reads for each DNA/RNA sample. There are many existing bioinformatics tools developed for the analysis and visualization of this data, but very large studies present computational and organizational challenges that are difficult to overcome manually. We designed a comprehensive pipeline for the analysis of RNA sequencing which leverages many existing tools and parallel computing technology to facilitate the analysis of extremely large studies. RNA CoMPASS provides a web-based graphical user interface and distributed computational pipeline including endogenous transcriptome quantification and additionally the investigation of exogenous sequences.

Species of Valerianaceae in South America represent one of the best examples of rapid diversification on a continental scale. The phylogeny of Valerianaceae has received a lot of attention within the last 10 years, but relationships among the South American species are fairly unresolved. Results from previous studies have not been well resolved with traditional genetic markers, most likely due to its recent and rapid radiation. Species in this clade exhibit a variety mating systems and inflorescence types. For the first part of this research I used several traditional plastid markers, and 3 new low copy nuclear markers to better resolve the phylogeny and then explore mating system evolution within the clade. For the second part of this research I collected high-throughput “next-generation” genomic sequence data from reduced representation libraries obtained using genotyping-by-sequencing (GBS) protocols, along with several phylogenetic methods, to try to further resolve the phylogeny of this group.

The relatively low cost of Next-Generation Sequencing (NGS) has enabled researchers to generate large amounts of sequencing data in order to identify disease-causing mutations and to assemble simple genomes. However, NGS has inherent limitations due to the short DNA read lengths and high error rate associated with the technique. The short read lengths of NGS prevent the assembly of genomes with long stretches of repetitive DNA, and the high error rate prevents the accurate detection of rare mutations in heterogeneous populations such as tumors and microbiomes. I have co-developed new NGS methods to overcome these challenges. In order to increase the effective read length of NGS reads, local de novo assembly of short reads into long contigs can be achieved through the use of Paired-End Restriction-site Associated DNA Sequencing (RAD-PE-Seq). With the RAD-PE method, I sequenced a stickleback fosmid and generated contigs with an N50 length of 480 nucleotides. In order to eliminate false-positive mutations caused by the high error rate of NGS, the Paired-End Low Error Sequencing (PELE-Seq) method was developed, which uses numerous quality control measures during the sequencing library preparation and data analysis steps in order to effectively eliminate sequencing errors. Control testing of the PELE-Seq demonstrates that the method completely eliminates false-positive mutations at sequencing read depths below 20,000X coverage, compared to a ~20% false-positive rate obtained with previous methods. The high accuracy of the PELE-Seq method allows for the detection of ultra-rare mutations in a genome, which was previously impossible with NGS. This dissertation includes previously published and unpublished co-authored material.

No description available.

Metagenomic sequencing is an increasingly popular way of determining microbial diversity from environmental and clinical samples. By specifically targeting the 16S rRNA gene found in all bacteria, classifications of pathogens can be determined based on the variable and conserved regions found in the gene. Metagenomic sequencing can therefore highlight the vast difference in microbiological diversity between culture-dependent and culture-independent methods. Today, this has expanded into various next-generation sequencing platforms which can provide massively parallel sequencing of the target fragment. One of these platforms is Ion-torrent, which can be utilized for targeting the 16S rRNA gene and with the help of bioinformatics pipelines be able to classify pathogens using the bacteria’s own variable and conserved regions. The overall aim of the present work is to evaluate the clinical use of Ion-torrent 16S ribosomal RNA sequencing for determining pathogenic species from clinical samples, but also to set up a pipeline for clinical practice. Optimal DNA-extraction and quantification methods were determined towards each evaluated sample-type and DNA-eluates were sent for 16S rRNA Sanger and Next-generation sequencing. The result indicated that the next-generation sequencing shows a concordance in results towards the culturing-based method, but also the importance of experimental design and effective quality trimming of the NGS data. The conclusion of the project is that the Ion-torrent pipeline provided by the Public Health Agency of Sweden shows great promise in determining pathogens from clinical samples. However, there is still a lot of validation and standardisations needed for the successful implementation into a clinical setting.

Deficiência intelectual (DI), associada ou não a outras alterações congênitas, é a razão mais frequente de procura por aconselhamento genético pelas famílias. Até alguns anos atrás, a realização de cariótipo, triagem para doenças metabólicas e fra(x) elucidavam apenas &sim;40% dos casos de pacientes com DI idiopática. Com o surgimento de arrays genômicos, as causas moleculares por trás de outros &sim;20% dos quadros de DI foram elucidadas; porém, mesmo com esse avanço, muitos pacientes ainda permanecem sem causa molecular clara que justifique o fenótipo. O sequenciamento do exoma (WES) é hoje um dos recursos disponíveis para o diagnóstico e possível elucidação das causas genéticas por trás da deficiência intelectual idiopática, abrindo caminho também à identificação de novos genes. O presente trabalho realizou o sequenciamento de exoma de 8 probandos que tinham em comum a deficiência intelectual esporádica, acompanhada ou não de outros sinais clínicos, e de seus genitores não afetados (trios). Esses pacientes foram previamente triados para a síndrome do X frágil, e submetidos a exame de array CGH para investigação de perdas e ganhos de segmentos cromossômicos, ambos com resultados negativos. O objetivo desse estudo foi detectar alterações e possivelmente novos genes associados com a DI, usando pipelines de padrões de herança mendeliano. Treze alterações em 9 genes foram detectadas por sequenciamento de exoma e confirmadas por sequenciamento Sanger: 8 mutações bialélicas em genes recessivos (TBC1D24, ADAMTSL2, NALCN, VPS13B), uma ligada ao X (MID1), e 4 alterações de novo (RYR2, GABBR2, CDK13, DDX3X); 5 dessas alterações ainda não haviam sido descritas nos bancos de dados consultados, caracterizando mutações novas. Dos 8 trios, em 5 identificamos alterações moleculares provavelmente responsáveis pelos quadros apresentados; dois desses casos foram em genes recessivos (mutações homozigotas ou em heterozigose composta) e potencialmente teriam sido detectados mesmo se apenas os probandos houvessem sido sequenciados. Para as alterações em heterozigose, porém, a avaliação dos genitores e constatação de status de novo da mutação foram importantes para avaliar o impacto da variante. Esse trabalho resultou em uma taxa de diagnóstico de 62,5%; mesmo considerando o pequeno tamanho da amostra, esse valor está bem acima dos 15-30% relatados na literatura quando essa metodologia é utilizada para o estudo de casos esporádicos de DI. Em dois casos, mutações foram identificadas em genes que só foram descritos como mutados recentemente e que ainda não são considerados genes de deficiência intelectual no OMIM: o gene CDK13 foi descrito como mutado em pacientes de uma única coorte com malformação cardíaca congênita (sindrômica ou não), porém sua contribuição para coortes de DI ainda não foi investigada. O gene GABBR2, identificado mutado em heterozigose em um dos nossos pacientes, já havia sido considerado um candidato potencial para DI, mas apenas 2 trabalhos detectaram mutações nesse gene entre pacientes com DI e epilepsia. Os resultados aqui apresentados substanciam o papel desses genes como implicados na DI sindrômica de herança autossômica dominante, e devem contribuir para serem considerados genes OMIM de deficiência intelectual / Intellectual disability (ID), associated or not with other congenital abnormalities, is the most frequent reason for families to seek genetic counseling. Until some years ago, karyotyping, metabolic disease and FRAXA screening elucidated only &sim;40% of patients with idiopathic ID. Importantly, with the introduction of genomic arrays, the molecular cause behind a further &sim;20% of ID cases was determined; however, despite this improvement, many patients are still not provided with a clear molecular explanation and cause for their phenotype. Nowadays, whole exome sequencing (WES) is one of the methods available for diagnosis and a further means of possible elucidation of the genetic causes of idiopathic intellectual disability; in many cases this method also allows identification of genes that have not been previously related to ID. In the present project, we sequenced the exome (WES) of 8 sporadic patients that all had ID, with or without other clinical signs, and their unaffected parents (trios); these patients had been previously screened for fragile X syndrome and for losses and gains of chromosomal segments by array CGH, both with negative results. The objective of this study was to detect mutations and possibly new genes associated with ID, using pipelines for Mendelian inheritance patterns. Thirteen mutations in 9candidate genes were detected by exome sequencing and confirmed by Sanger sequencing, among them 8 biallelic mutations in autossomal recessive genes (TBC1D24, ADAMTSL2, NALCN, VPS13B), one mutation in an X-linked gene (MID1), and 4 de novo alterations (RYR2, GABBR2, CDK13, DDX3X); 5 of these mutations had not been described in the databases consulted characterizing new variants. Of the 8 trios, we obtained a probable diagnosis of the molecular alteration responsible for the presented phenotypes in 5. Two of these cases were in recessive genes (homozygous mutations or compound heterozygous), and the mutations would probably have been detected even if only the probands had been sequenced. However, for the heterozygous mutations, the assessment of the parents and the confirmation of the de novo status of the mutation was important to evaluate the impact of the variant. This work resulted in a diagnosis rate of 62.5%; even considering the small sample size, this value is well above the average of 15-30% reported in the literature when the methodology used for the study of ID sporadic cases is considered. In two cases, mutations were detected in genes only recently described as mutated and which are not considered yet as OMIM ID genes. The CDK13 gene had already been described as mutated in a single cohort of patients with syndromic congenital heart defects, but its contribution to ID cohorts has not been established. The GABBR2 gene, where a heterozygous mutation was identified in the patient, had already been considered a potential candidate for ID; there are only 2 studies that detected mutations in this gene among patients with ID and epilepsy. This contribution may pave the way to establishing GABBR2 and CDK13 as causations of ID and acceptance by OMIM