Bioinformatic approaches for genome finishing

Husemann P, Tauch A. Bioinformatic approaches for genome finishing. Bielefeld: Universitätsbibliothek Bielefeld; 2011

The porcine intestinal microbiota : studies on diversity and dietary impact

The entirety of microbial communities within the gastrointestinal tract is referred to as intestinal microbiota and is predominantly composed of bacteria. Interactions between the microbiota, the host and the diet are essential for maintaining a healthy and functional intestinal ecosystem. The overarching aim of this thesis was the characterization of the porcine intestinal microbiota and further to enhance knowledge about the effects of varying diets. High-throughput sequencing of the 16S rRNA gene facilitates exploration of the taxonomic composition of the microbiota. However, the respective findings may be impaired by methodological variations. Thus, within this thesis, commercial DNA extraction kits are evaluated for their suitability in porcine microbiota analysis. The tested extractions yield into variations of quantity and quality of DNA. The DNA extracts are further used to elucidate the structure of the microbiota by a rapid fingerprinting (Terminal Restriction Fragment Length Polymorphism) and high-resolution sequencing (Illumina amplicon sequencing). While different variable regions of the 16S rRNA gene vary in the taxonomical resolution, sequencing analyses exhibit a good comparability of the two regions V1-V2 and the V5-V6. Furthermore, the microbiota profiles reveal a high consistency by the fingerprinting and sequencing approach but are distinguished by the different DNA extraction kits. Based on criteria of DNA extraction and the depicted microbiota composition, it is recommended to use the FastDNA SPIN Kit for Soil for further analysis of porcine intestinal microbiota. Subsequently, these methodological findings are applied to investigate the impact of varying diets. Illumina amplicon sequencing of the V1-V2 region of the 16S rRNA gene reveals different microbiota structures when diets are solely composed of rye or triticale. Besides the taxonomic analyses of ileal digesta and fecal samples, the concentrations of bacterial metabolites in feces are determined. In summary, rye promotes an increased abundance of saccharolytic bacteria like Lactobacillus, Bifidobacterium, and Prevotella and results in higher concentrations of bacterial metabolites in fecal samples. In contrast, a diet based on triticale is associated with an increased abundance of Clostridium sensu stricto, which may indicate an enhanced cellulolytic potential of the microbiota. When the crude protein content is increased (18%), compared to a lower content (14%), an increased abundance of Lactobacillus is demonstrated in microbiota of ileal digesta samples. However, the content of crude protein did not affect the overall microbiota significantly. In addition, dietary supplementation with probiotic Bacillus spp. shows no effect. In conclusion, these dietary effects on microbiota are considered together with results of a protein digestibility analysis. Moreover, an impact of dietary calcium and phosphorus in combination with different sources of dietary protein is analyzed by fingerprinting approach of digesta samples. Here, the content of calcium-phosphorus shows significant effects on the microbiota of caecal digesta and the putative identities of discriminative variables are determined by a cloning-sequencing approach. Similar, 16S rRNA gene sequencing reveals a significant impact of dietary calcium-phosphorus on the overall fecal microbiota without indicating specific discriminating variables. In combination with the results of a meta-proteomic approach, a gradual adaptation on dietary changes is indicated and consequently, a prolonged adaptation time of three to four weeks is recommended for diet-microbiota studies. This thesis includes a comprehensive analysis of the microbiota across and along the gastrointestinal tract of piglets and explores the dietary inclusion of four levels of insect larvae meal. Feeding insects represent an alternative source of dietary protein, whereby the increased content of chitin indicates a potential shift in microbiota composition compared to a control diet. However, in this case, the structural analysis demonstrates no effects on the overall microbiotas structure. However, a pairwise comparison between diets reveals significant effects on the microbiota of digesta samples of the small intestine. Dietary inclusion of 5% insect meal increases the abundance of Lactobacillus, whereas the control treatment promotes Bifidobacterium. In conclusion, the results of the present thesis emphasize the importance of standardization within 16S rRNA gene based studies of the porcine intestinal microbiota. Furthermore, the necessity of studying various sampling sites combined with multidisciplinary approaches is demonstrated.Die Gesamtheit der mikrobiellen Gemeinschaften im Gastrointestinaltrakt wird als intestinale Mikrobiota bezeichnet und setzt sich vorwiegend aus Bakterien zusammen. Wechselwirkungen zwischen der Mikrobiota, dem Wirt und der Ernährung sind unerlässlich für ein gesundes und funktionelles Ökosystem im Darm. Das übergeordnete Ziel dieser Thesis war die Charakterisierung der intestinalen Mikrobiota des Schweines und Kenntnisse über die Auswirkungen unterschiedlicher Diäten zu erweitern. Hochdurchsatz-Sequenzierung des 16S rRNA Gens erleichtert die Erforschung der taxonomischen Zusammensetzung der Mikrobiota. Allerdings können die jeweiligen Ergebnisse durch methodische Abweichungen beeinträchtigt sein. Aufgrund dessen werden im Rahmen dieser Thesis kommerzielle DNA Extraktions-Kits für ihre Eignung in der Analyse der Schweine-Mikrobiota bewertet. Die getesteten Extraktionen bringen Unterschiede in der DNA Quantität und Qualität hervor. Im Weiteren wird die DNA zur Aufklärung der Mikrobiota-Struktur, durch eine schnelle Fingerprinting-Methode (Terminales Restriktionsfragmentlängen-polymorphismus) und einem hochauflösenden Sequenzierungsansatz (Illumina Amplikon Sequenzierung), eingesetzt. Zwar variieren unterschiedliche variable Regionen des 16S rRNA Gens in der taxonomischen Auflösung, doch belegen Sequenzierungsanalysen eine gute Vergleichbarkeit von V1-V2 und V5-V6. Des Weiteren offenbaren die Mikrobiota-Profile eine hohe Übereinstimmung zwischen Fingerprinting und Sequenzierung, unterscheiden sich aber zwischen den unterschiedlichen DNA Extraktions-Kits. Basierend auf Kriterien der DNA Extraktion und der aufgeklärten Mikrobiota-Zusammensetzung, wird das FastDNA SPIN Kit for Soil für weitere Analysen der intestinalen Mikrobiota des Schweines empfohlen. Diese methodischen Erkenntnisse werden anschließend angewendet, um die Auswirkungen durch verschiedene Diäten zu untersuchen. So zeigt Illumina Amplikon Sequenzierung der V1-V2 Region des 16S rRNA Gens unterschiedliche Mikrobiotastrukturen auf, wenn Diäten sich nur aus Roggen oder Triticale zusammensetzen. Neben der taxonomischen Analyse von Chymusproben aus dem Ileum und dem Kot, werden auch Konzentrationen von bakteriellen Metaboliten bestimmt. Zusammenfassend fördert Roggen eine erhöhte Abundanz von saccharolytischen Bakterien wie Lactobacillus, Bifidobacterium und Prevotella und führt zu höheren Konzentrationen von bakteriellen Metaboliten im Kot. Im Gegensatz dazu ist eine Triticale-Diät mit einer erhöhten Abundanz von Clostridium sensu stricto assoziert, was ein erhöhtes cellulolytisches Potential der Mikrobiota andeutet. Bei einem erhöhtem Rohproteingehalt (18%) in der Diät, im Vergleich zu einem geringeren Gehalt (14%), wird eine erhöhte Lactobacillus-Abundanz im Chymus des Ileums aufgezeigt. Der Gehalt des Rohproteins beeinflusst allerdings die Gesamtheit der Mikrobiota nicht signifikant. Auch zeigt die diätische Ergänzung von probiotischen Bacillus spp. keinen Effekt. Abschließend werden diese diätischen Effekte auf die Mikrobiota gemeinsam mit den Ergebnissen der Proteinverdaulichkeitsanalyse betrachtet. Im Weiteren wird, anhand eines Fingerprinting-Ansatzes, ein Einfluss von Calcium und Phosphat in Kombination mit verschiedenen Proteinquellen in Chymusproben untersucht. Hier zeigt der Calcium-Phosphat Gehalt einen signifikanten Effekt auf die Mikrobiota im Chymus des Blinddarms. Die dafür verantwortlichen Bakterien werden durch einen Klonierung-Sequenzierung-Ansatz identifiziert. Ähnlich hierzu, weist die 16S rRNA Gen Sequenzierung von Kotproben einen signifikanten Einfluss des diätischen Calcium-Phosphors auf, ohne dabei auf bestimmte beeinflusste Bakterien hin zu deuten. Gemeinsam mit Ergebnissen eines Meta-Proteomik Ansatzes wird auf eine graduelle Anpassung an diätische Veränderungen hingewiesen und daher wird eine verlängerte Adaptationszeit von drei bis vier Wochen für Diät-Mikrobiota Studien empfohlen. Diese Thesis enthält auch eine umfassende Analyse der Mikrobiota quer durch und entlang des Gastrointestinaltraktes von Ferkeln und untersucht hier vier verschiedene Stufen einer Rationszulage von Insektenlarvenmehl. Die Verfütterung von Insekten stellt eine alternative Quelle von Protein dar, wobei, im Vergleich zu einer Kontrolldiät, der erhöhte Chitingehalt auf eine potentielle Veränderung der Mikrobiota hinweist. Hier zeigt allerdings nur ein paarweiser Vergleich zwischen den Diäten einen signifikanten Einfluss auf die Mikrobiota in Chymus Proben aus dem Dünndarm. Diätische Zulage von 5% Insektenmehl erhöht die Abundanz von Lactobacillus, wohingegen die Kontrollgruppe Bifidobacterium fördert. Abschließend heben die Ergebnisse dieser Thesis die Bedeutung einer Standardisierung innerhalb 16S rRNA Gen basierten Studien der intestinalen Mikrobiota des Schweines hervor. Des Weiteren wird die Notwendigkeit der Untersuchung verschiedener Probenentnahmestellen zusammen mit dem Einsatz von interdisziplinären Ansätzen aufgezeigt

Computational methods for functional analysis of plant small RNAs using the RNA degradome

Small RNAs (sRNAs) are a broad class of short regulatory non-coding RNAs that play critical roles in many important biological pathways. They suppress the translation of messenger RNAs (mRNAs) by directing the RNA-induced silencing complex to their sequence-specific mRNA target(s). In plants, this typically results in mRNA cleavage and subsequent degradation of the mRNA. Cleaved mRNA frag­ments can be captured on a genome-wide scale using a high-throughput sequencing technique called degradome sequencing, which can then be used to identify causal sRNAs. Recent improvements to sequencing technologies have resulted in typical se­quencing experiments now producing millions of unique reads. This has led to new challenges in bioinformatics regarding the computation time and resources required to perform sRNA and degradome data analyses. In this thesis, we present three new sRNA and degradome analysis tools that we have developed called PAREsnip2, PAREameters and NATpare. PAREsnip2 is a tool we developed to predict sRNA targets, on a genome-wide scale, using degradome data and configurable targeting rules. Employing novel sequencing encoding and data structures, PAREsnip2 outperforms existing tools in computation time, at times by more than two orders of magnitude, with minimal computational resource requirements. PAREameters is a computational method for inference of plant microRNA targeting rules, using the degradome, that can then be employed by PAREsnip2. Benchmarking on multiple A. thaliana datasets show that the computationally inferred criteria outperform currently used criteria in terms of sensitivity on all datasets while maintaining precision on most. NATpare is a tool for high-throughput prediction and functional analysis of nat-siRNAs using the degradome. NATpare is the first tool of its kind to combine nat-siRNA prediction with functional analysis using the degradome. Compared to current methods, our new algorithm speeds up computation time by over two orders of magnitude when analysing an A thaliana dataset. We also demonstrate that it is the only computational method able to complete analyses of non-model organisms within a reasonable time frame. We exemplify the use of these computational methods by performing functional analysis of CMV D-satRNA derived sRNA in S. lycopersicum to better understand their role in virus induced plant death

Genomics and transcriptomics of the Mycobacterium tuberculosis complex

The goal of eliminating tuberculosis (TB) by 2050 depends on the development of improved TB diagnostics, drugs and vaccines. Advances in these areas require a deep understanding of the disease and its causative agent, Mycobacterium tuberculosis (M. tb). Mycobacterial species that cause TB in humans and other mammalian hosts are grouped within the M. tb complex. Development of powerful technologies such as next-generation sequencing and microarrays opened up new avenues for comparative and functional genomics of the M. tb complex. Due to the large and increasingly complex datasets generated from these technologies, the bottleneck in biological investigation has shifted from data generation to analysis. The objectives of this thesis were to establish and employ strategies for the analysis, integration, and interpretation of high-throughput sequencing and microarray datasets using a range of bioinformatics and statistical tools. In the area of comparative genomics, we assessed the genetic diversity in the M. tb complex using various methods, such as SNP (single nucleotide polymorphism) genotyping, automated Sanger sequencing and next-generation sequencing. In a study comparing the genomes of the virulent M. bovis and M. bovis BCG vaccine strains, we identified a set of SNPs that were common to all BCG strains, and could provide novel insights on the molecular basis of BCG attenuation. In another study, we surveyed the genetic variation in the highly immunodominant esx gene family among clinical isolates of M. tb and identified sequence polymorphisms in known T- cell epitopes on Esx proteins that could affect their immunogenicity. We exploited the power of next-generation sequencing to detect sequence variation among M. tb strains that could result in phenotypic differences. By comparing the genomes of drug-resistant mutants with the sensitive wild-type strain we were able to identify the target of the anti-TB drug, pyridomycin. Using a similar approach we identified a mutation that makes M. tb strains incapable of producing PDIMs (phthiocerol dimycocerosates), which are cell wall associated lipids involved in M. tb virulence. In the area of functional genomics, we mapped genome-wide binding sites for transcription factors using chromatin immunoprecipitation followed by hybridization to microarrays (ChIP-on-chip) or sequencing (ChIP-seq), and performed transcription profiling by means of high-throughput cDNA sequencing (RNA-seq). We carried out a comprehensive study to characterize the whole transcriptome of M. tb in exponential and stationary phases of growth, and understand the genome-wide dynamics of two key components of the transcription machinery, namely, RNA polymerase and NusA. By systematic integration of the ChIP-seq and RNA-seq data, we identified a set of transcription units (TU) in the M. tb genome, and mapped their putative promoters. Analysis of RNAP and NusA binding across the promoter and body of TUs and their correlation with transcription uncovered new functional aspects of the transcriptional complex in M. tb. We also exploited the ChIP-on-chip and ChIP-seq technologies to define the regulon of the M. tb sigma factor F, and gain a better understanding of the regulatory role of the nucleoid associated protein, EspR. Altogether, this thesis has improved our knowledge of the evolution, physiology and virulence of the M. tb complex. In addition, we have established next generation sequencing as a powerful tool for comparative and functional studies, with potential applications in the clinical setting

Going viral : an integrated view on virological data analysis from basic research to clinical applications

Viruses are of considerable interest for several fields of life science research. The genomic richness of these entities, their environmen- tal abundance, as well as their high adaptability and, potentially, pathogenicity make treatment of viral diseases challenging. This thesis proposes three novel contributions to antiviral research that each concern analysis procedures of high-throughput experimen- tal genomics data. First, a sensitive approach for detecting viral genomes and transcripts in sequencing data of human cancers is presented that improves upon prior approaches by allowing de- tection of viral nucleotide sequences that consist of human-viral homologs or are diverged from known reference sequences. Sec- ond, a computational method for inferring physical protein contacts from experimental protein complex purification assays is put for- ward that allows statistically meaningful integration of multiple data sets and is able to infer protein contacts of transiently binding protein classes such as kinases and molecular chaperones. Third, an investigation of minute changes in viral genomic populations upon treatment of patients with the mutagen ribavirin is presented that first characterizes the mutagenic effect of this drug on the hepatitis C virus based on deep sequencing data.Viren sind von beträchtlichem Interesse für die biowissenschaftliche Forschung. Der genetische Reichtum, die hohe Vielfalt, wie auch die Anpassungsfähigkeit und mögliche Pathogenität dieser Organismen erschwert die Behandlung von viralen Erkrankungen. Diese Promotionsschrift enthält drei neuartige Beiträge zur antiviralen Forschung welche die Analyse von experimentellen Hochdurchsatzdaten der Genomik betreffen: erstens, ein sensitiver Ansatz zur Entdeckung viraler Genome und Transkripte in Sequenzdaten humaner Karzinome, der die Identifikation von viralen Nukleotidsequenzen ermöglicht, die von Referenzgenomen ab- weichen oder homolog zu humanen Faktoren sind. Zweitens, eine computergestützte Methode um physische Proteinkontakte von experimentellen Proteinkomplex-Purifikationsdaten abzuleiten welche die statistische Integration von mehreren Datensätzen erlaubt um insbesondere Proteinkontakte von flüchtig interagierenden Proteinklassen wie etwa Kinasen und Chaperonen aus den Daten ableiten zu können. Drittens, eine Untersuchung von kleinsten Änderungen viraler Genompopulationen während der Behandlung von Patienten mit dem Mutagen ribavirin die zum ersten Mal die mutagene Wirkung dieses Medikaments auf das Hepatitis C Virus mittels Tiefensequenzdaten nachweist

Annotated Cell and Molecular Biology 5e: What We Know and How We Found Out

https://dc.uwm.edu/biosci_facbooks_bergtrom/1013/thumbnail.jp

Basic Cell and Molecular Biology 5e: What We Know and How We Find Out

https://dc.uwm.edu/biosci_facbooks_bergtrom/1014/thumbnail.jp