Computational Methods for Integrative Structural Variant Analysis Across Species Boundaries

Structural variations (SVs) are a phenomenon that have a tremendous impact on all species. SVs are the result of fundamental rearrangement mechanisms but can lead to severe human diseases like cancer. Rearrangement events also provide means that enable bacteria to adapt to environmental pressures where they can also happen across species boundaries in events called horizontal gene transfer (HGT). The incorporation of foreign genes from a donor into an acceptor genome can be investigated on the genomic level, the activity and protein expression changes, however, are better revealed on the proteomic level. This thesis contributes four computational methods for the detection of complex SVs of various types and sizes including HGT events from genomic next-generation sequencing (NGS) data and proteomic shotgun mass-spectrometry (MS) data. Concerning HGT events, our methods address the questions of what organisms are involved in the transfer, what genes are exactly transferred and to what position, and what are the implications on proteomic level. First, we present the generic SV detection tool Gustaf. Gustaf improves the size and type resolution compared to previous SV detection methods. A further specific advantage is the characterisation of translocations and dispersed duplications as a combination of simple, delocalised variants that have to be inferred from separate SV calls. With this basis for a more in-depth focus on HGT detection, we developed two mapping-based methods, Daisy and DaisyGPS. Daisy facilitates Gustaf and further SV detection strategies to precisely identify the transferred region within the donor and its insertion site in the acceptor genome. DaisyGPS uses metagenomic profiling strategies to identify suitable acceptor and donor references. In contrast to previous approaches based on sequence composition patterns or phylogenetic disagreements, our methods provide a detection based on sequence comparison and hence offer novel means of evidence. In the last project, we present a method for HGT detection, called Hortense, that is based on proteomic MS data. Hortense extends a standard database peptide search with a thorough cross-validation to ensure HGT properties, and is the first dedicated proteomics HGT detection method. Results from Hortense can also serve as supporting evidence and functional confirmation for HGT events proposed by our genomic-based methods. Taken together, the three HGT methods provide a full view of the transfer event that was not be possible before or with one of the methods alone.Strukturvariationen (SVs) haben eine immense Bedeutung im Genom sämtlicher Spezies. Sie sind das Ergebnis fundamentaler Rekonstruktionsmechanismen und verleihen gleichzeitig Bakterien die Fähigkeit, sich an ihre Umgebung anzupassen. In Bakterien gibt es zudem das Phänomen des horizontalen Gentransfers (HGT), bei dem Gene über Speziesgrenzen hinweg von einem Donor-Individuum zu einem anderen Akzeptor übertragen werden. Die Integration eines neuen Gens kann auf genomischer Ebene untersucht werden. Die Aktivität und Expression hingegen lässt sich nur auf Proteinebene bestimmen. In dieser Doktorarbeit werden bioinformatische Methoden zur Detektion von komplexen SVs unterschiedlichen Typs und Größe anhand von Next- generation Se- quencing Daten und proteomischen Massenspektrometriedaten mit einem Fokus auf HGT-Events vorgestellt. Bei einem HGT-Event muss zunächst bestimmt werden, zwischen welchen Organismen der Transfer stattgefunden hat und welche Gene aus dem Donor an welcher Stelle im Akzeptor eingefügt wurden. Anschließend kann man untersuchen, ob das transferierte potentielle Protein auch funktionell ist. Als erstes wird das SV-Detektionstool Gustaf vorgestellt, welches eine bessere Auflösung bezogen auf Größe und Typ von SVs im Vergleich zu vorherigen Methoden ermöglicht. Einen besonderen Vorteil bietet Gustaf in der Charakterisierung von komplexen Translokationen und Duplikationen als Kombination von simpleren, im Genom voneinander entfernten Varianten. Mit dieser generischen Methode als Basis wurden zwei mapping-basierte Methoden, Daisy und DaisyGPS, zur HGT-Detektion entwickelt. Daisy verwendet Gustaf und weitere SV-Detektionsstrategien um die transferierte Region im Donorgenom und ihre Insertionsstelle im Akzeptorgenom präzise zu bestimmen. DaisyGPS verwendet etablierte Strategien für die metagenomische Bestimmung von Mikroorganismen in einer Probe, um eine passende Akzeptor- und Donorreferenz zu identifizieren. Daisy und DaisyGPS basieren auf Sequenzvergleichen und heben sich damit von den bisher existierenden Methoden ab, welche HGTs anhand von Sequenzkompositionsmustern und phylogenetischen Inkonsistenzen bestimmen. Im letzten Projekt wird die proteomische Methode Hortense vorgestellt. Hortense erweitert die Standarddatenbanksuche von Spektren um eine umfassende Kreuzvalidierung, um definierte Eigenschaften eines HGT-Proteins sicher zu stellen. Alle drei Methoden zur HGT-Detektion ermöglichen eine ganzheitliche Analyse von HGT-Events, welche vorher oder nur mit einer einzelnen der drei Methoden nicht möglich wäre

Systems-level analysis reveals multiple modulators of epithelial-mesenchymal transition and identifies DNAJB4 and CD81 as novel metastasis inducers in breast cancer

Epithelial-mesenchymal transition (EMT) is driven by complex signaling events that induce dramatic biochemical and morphological changes whereby epithelial cells are converted into cancer cells. However, the underlying molecular mechanisms remain elusive. Here, we used mass spectrometry based quantitative proteomics approach to systematically analyze the post-translational biochemical changes that drive differentiation of human mammary epithelial (HMLE) cells into mesenchymal. We identified 314 proteins out of more than 6,000 unique proteins and 871 phosphopeptides out of more than 7,000 unique phosphopeptides as differentially regulated. We found that phosphoproteome is more unstable and prone to changes during EMT compared with the proteome and multiple alterations at proteome level are not thoroughly represented by transcriptional data highlighting the necessity of proteome level analysis. We discovered cell state specific signaling pathways, such as Hippo, sphingolipid signaling, and unfolded protein response (UPR) by modeling the networks of regulated proteins and potential kinase-substrate groups. We identified two novel factors for EMT whose expression increased on EMT induction: DnaJ heat shock protein family (Hsp40) member B4 (DNAJB4) and cluster of differentiation 81 (CD81). Suppression of DNAJB4 or CD81 in mesenchymal breast cancer cells resulted in decreased cell migration in vitro and led to reduced primary tumor growth, extravasation, and lung metastasis in vivo. Overall, we performed the global proteomic and phosphoproteomic analyses of EMT, identified and validated new mRNA and/ or protein level modulators of EMT. This work also provides a unique platform and resource for future studies focusing on metastasis and drug resistanceTurkiye Cumhuriyeti Kalkinma Bakanlig

Synopse virologischer Analysen im Nationalen Referenzzentrum für Influenzaviren während der COVID-19-Pandemie

Das Nationale Referenzzentrum für Influenzaviren gewinnt durch die fortlaufende Untersuchung von Proben aus den Sentinelpraxen der Arbeitsgemeinschaft Influenza einen umfassenden Überblick über die zirkulierenden respiratorischen Erreger in Deutschland. Dazu gehören neben SARS-CoV-2 und den Influenzaviren auch das Respiratorische Synzytialvirus, Parainfluenzaviren, humane Metapneumoviren, humane saisonale Coronaviren und humane Rhinoviren. Die Analyseergebnisse von 15.660 Sentinelproben sowie weiteren Isolaten im Zeitraum von Kalenderwoche 5/2020 bis 21/2022 werden im Epidemiologischen Bulletin 22/2022 vorgestellt. Beschrieben werden außerdem die Zirkulation respiratorischer Erreger im Vergleich zu vorpandemischen Saisons, die molekulare Charakterisierung und phylogenetische Analysen, die Überprüfung der Passgenauigkeit der eingesetzten Influenzaimpfstoffe und die Resistenzprüfung von Influenzaviren

Somatic Mutation Profiles of MSI and MSS Colorectal Cancer Identified by Whole Exome Next Generation Sequencing and Bioinformatics Analysis

BACKGROUND: Colorectal cancer (CRC) is with approximately 1 million cases the third most common cancer worldwide. Extensive research is ongoing to decipher the underlying genetic patterns with the hope to improve early cancer diagnosis and treatment. In this direction, the recent progress in next generation sequencing technologies has revolutionized the field of cancer genomics. However, one caveat of these studies remains the large amount of genetic variations identified and their interpretation. METHODOLOGY/PRINCIPAL FINDINGS: Here we present the first work on whole exome NGS of primary colon cancers. We performed 454 whole exome pyrosequencing of tumor as well as adjacent not affected normal colonic tissue from microsatellite stable (MSS) and microsatellite instable (MSI) colon cancer patients and identified more than 50,000 small nucleotide variations for each tissue. According to predictions based on MSS and MSI pathomechanisms we identified eight times more somatic non-synonymous variations in MSI cancers than in MSS and we were able to reproduce the result in four additional CRCs. Our bioinformatics filtering approach narrowed down the rate of most significant mutations to 359 for MSI and 45 for MSS CRCs with predicted altered protein functions. In both CRCs, MSI and MSS, we found somatic mutations in the intracellular kinase domain of bone morphogenetic protein receptor 1A, BMPR1A, a gene where so far germline mutations are associated with juvenile polyposis syndrome, and show that the mutations functionally impair the protein function. CONCLUSIONS/SIGNIFICANCE: We conclude that with deep sequencing of tumor exomes one may be able to predict the microsatellite status of CRC and in addition identify potentially clinically relevant mutations

Intergenerational transmission of fertility timing in Germany

<b>Background</b>: Intergenerational transmission of completed fertility is widely confirmed for several societies. Less research, however, has focused on differences in the transmission effect of fertility timing and its underlying mechanisms in a regional context. <b>Objective</b>: The aim of this study is to examine the association between a mother's age at her daughter's birth and that daughter's transition to first birth in eastern and western Germany, as well as its underlying mechanisms. <b>Methods</b>: Using data from the German Family Panel (pairfam), the intergenerational transmission of fertility timing between mothers and daughters born between 1971-1973 and 1981-1983 is investigated using event history analysis. As an alternative to a mother's age at first birth, a mother's age at her daughter's birth is used to determine her daughter's transition to first birth. <b>Results</b>: Results show evidence for intergenerational transmission of young childbearing between mothers and their daughters in eastern and western Germany, though the association was weaker for eastern Germany. This intergenerational transmission effect cannot be explained by the measures used to capture the underlying mechanisms - socialisation, socioeconomic status transmission, and social control. <b>Contribution</b>: Our contribution to the ongoing discussion is to close a gap in research on the intergenerational transmission of fertility timing. By using the German context to analyse regional differences, we exemplify the varying strength of the intergenerational transmission of fertility timing between eastern and western Germany that persisted beyond reunification

Where did you come from, where did you go: Refining metagenomic analysis tools for horizontal gene transfer characterisation

Horizontal gene transfer (HGT) has changed the way we regard evolution. Instead of waiting for the next generation to establish new traits, especially bacteria are able to take a shortcut via HGT that enables them to pass on genes from one individual to another, even across species boundaries. The tool Daisy offers the first HGT detection approach based on read mapping that provides complementary evidence compared to existing methods. However, Daisy relies on the acceptor and donor organism involved in the HGT being known. We introduce DaisyGPS, a mapping-based pipeline that is able to identify acceptor and donor reference candidates of an HGT event based on sequencing reads. Acceptor and donor identification is akin to species identification in metagenomic samples based on sequencing reads, a problem addressed by metagenomic profiling tools. However, acceptor and donor references have certain properties such that these methods cannot be directly applied. DaisyGPS uses MicrobeGPS, a metagenomic profiling tool tailored towards estimating the genomic distance between organisms in the sample and the reference database. We enhance the underlying scoring system of MicrobeGPS to account for the sequence patterns in terms of mapping coverage of an acceptor and donor involved in an HGT event, and report a ranked list of reference candidates. These candidates can then be further evaluated by tools like Daisy to establish HGT regions. We successfully validated our approach on both simulated and real data, and show its benefits in an investigation of an outbreak involving Methicillin-resistant Staphylococcus aureus data. Author summary Evolution is traditionally viewed as a process where changes are only vertically inherited from parent to offspring across generations. Many principles such as phylogenetic trees and even the “tree of life” are based on that doctrine. The concept of horizontal gene transfer changed the way we regard evolution completely. Horizontal gene transfer is the movement of genetic information between distantly related organisms of the same generation. Genome sequencing not only provided further evidence complementing experimental evidence but also shed light onto the frequency and prominence of this concept. Especially the rapid spread of antimicrobial resistance genes is a prominent example for the impact that horizontal gene transfer can have for public health. Next generation sequencing brought means for quick and relatively cheap analysis of even complex metagenomic samples where horizontal gene transfer is bound to happen frequently. Methods to directly detect and characterise horizontal gene transfer from such sequencing data, however, are still lacking. We here provide a method to identify organisms potentially involved in horizontal gene transfer events to be used in downstream analysis that enables a characterisation of a horizontal gene transfer event in terms of impact and prevalence

„Gefahrenhinweiskarte Schwäbisch-Fränkischer Jura“ – Flächendeckende Erfassung historischer Informationen zu Sturz- und Rutschprozessen, sowie zu Erdfällen

Naturereignisse wie Sturz- und Rutschungsprozesse sowie Erdfälle stellen in besiedelten Gebieten für den Menschen schon immer eine schwer einzuschätzende Bedrohung dar. In Deutschland sind nicht nur die Alpen als Hochgebirge betroffen, sondern auch in den Mittelgebirgen spielen Georisiken eine wichtige Rolle. Die Datenbasis über Ausmaß und Häufigkeit von Ereignissen und damit die Möglichkeit zur qualifizierten Beurteilung der Gefahrenlage ist bisher außerhalb des Alpenraums noch nicht ausreichend entwickelt. Da systematische Untersuchungen zu Naturgefahren in der Vergangenheit fehlen, können auch mögliche Veränderungen in der Prozessdynamik als Folge aktueller Klimaänderungen noch nicht ausreichend beurteilt werden. Um frühzeitig Antworten auf die anstehenden Fragen auch für die betroffene Bevölkerung geben zu können, soll das Thema Naturgefahren im außeralpinen Raum stärker in den Fokus der Forschung gerückt werden. Gefahrenhinweiskarten können hier im Bereich der Risikokommunikation ansetzen und bestehende Informationen über bisher stattgefundene Ereignisse und potentiell gefährdete Bereiche vermitteln. Im August 2010 wurde das Projekt „Gefahrenhinweiskarte Schwäbisch-Fränkischer Jura“ vom Bayerischen Landesamt für Umwelt, kofinanziert durch die EU, gestartet. Der Lehrstuhl für Physische Geographie der Katholischen Universität Eichstätt-Ingolstadt hat seit Oktober 2011 innerhalb dieses Projekts die flächendeckende historische Recherche von gravitativen Massenbewegungen und Erdfallereignissen für 152 Gemeinden und Städte im Fränkischen Jura übernommen. Über verschiedene Informationsquellen wie Gemeinde- und Zeitungsarchive, Zeitzeugen und behördliche Quellen werden zurückliegende Ereignisse seit etwa 1900 ermittelt. Die Aufnahme historischer und aktueller Ereignisse stellt wichtige Grundlagen für die Erstellung von Gefahrenhinweiskarten, regionale Spezialuntersuchungen (Gutachten) und Gefahrenmodellierungen zur Verfügung