Objective review of de novo stand-alone error correction methods for NGS data

[EN] The sequencing market has increased steadily over the last few years, with different approaches to read DNA information prone to different types of errors. Multiple studies demonstrated the impact of sequencing errors on different applications of next-generation sequencing (NGS), making error correction a fundamental initial step. Different methods in the literature use different approaches and fit different types of problems. We analyzed 50 methods divided into five main approaches (k-spectrum, suffix arrays, multiple-sequence alignment, read clustering, and probabilistic models). They are not published as a part of a suite (stand-alone), and target raw, unprocessed data without an existing reference genome (de novo). These correctors handle one or more sequencing technologies using the same or different approaches. They face general challenges (sometimes with specific traits for specific technologies) such as repetitive regions, uncalled bases, and ploidy. Even assessing their performance is a challenge in itself because of the approach taken by various authors, the unknown factor (de novo), and the behavior of the third-party tools employed in the benchmarks. This study aims to help the researcher in the field to advance the field of error correction, the educator to have a brief but comprehensive companion, and the bioinformatician to choose the right tool for the right job. © 2016 John Wiley & Sons, LtdWe want to thank our colleague Eloy Romero Alcale who has provided valuable advice regarding the structure of the document. This work was supported by Generalitat Valenciana [GRISOLIA/2013/013 to A.A.].Alic, AS.; Ruzafa, D.; Dopazo, J.; Blanquer Espert, I. (2016). Objective review of de novo stand-alone error correction methods for NGS data. Wiley Interdisciplinary Reviews: Computational Molecular Science. 6(2):111-146. https://doi.org/10.1002/wcms.1239S1111466

Bioinformatics for personal genomics: development and application of bioinformatic procedures for the analysis of genomic data

In the last decade, the huge decreasing of sequencing cost due to the development of high-throughput technologies completely changed the way for approaching the genetic problems. In particular, whole exome and whole genome sequencing are contributing to the extraordinary progress in the study of human variants opening up new perspectives in personalized medicine. Being a relatively new and fast developing field, appropriate tools and specialized knowledge are required for an efficient data production and analysis. In line with the times, in 2014, the University of Padua funded the BioInfoGen Strategic Project with the goal of developing technology and expertise in bioinformatics and molecular biology applied to personal genomics. The aim of my PhD was to contribute to this challenge by implementing a series of innovative tools and by applying them for investigating and possibly solving the case studies included into the project. I firstly developed an automated pipeline for dealing with Illumina data, able to sequentially perform each step necessary for passing from raw reads to somatic or germline variant detection. The system performance has been tested by means of internal controls and by its application on a cohort of patients affected by gastric cancer, obtaining interesting results. Once variants are called, they have to be annotated in order to define their properties such as the position at transcript and protein level, the impact on protein sequence, the pathogenicity and more. As most of the publicly available annotators were affected by systematic errors causing a low consistency in the final annotation, I implemented VarPred, a new tool for variant annotation, which guarantees the best accuracy (>99%) compared to the state-of-the-art programs, showing also good processing times. To make easy the use of VarPred, I equipped it with an intuitive web interface, that allows not only a graphical result evaluation, but also a simple filtration strategy. Furthermore, for a valuable user-driven prioritization of human genetic variations, I developed QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. The prioritization is achieved by a global positive selection process that promotes the emergence of the most reliable variants, rather than filtering out those not satisfying the applied criteria. QueryOR has been used to analyze the two case studies framed within the BioInfoGen project. In particular, it allowed to detect causative variants in patients affected by lysosomal storage diseases, highlighting also the efficacy of the designed sequencing panel. On the other hand, QueryOR simplified the recognition of LRP2 gene as possible candidate to explain such subjects with a Dent disease-like phenotype, but with no mutation in the previously identified disease-associated genes, CLCN5 and OCRL. As final corollary, an extensive analysis over recurrent exome variants was performed, showing that their origin can be mainly explained by inaccuracies in the reference genome, including misassembled regions and uncorrected bases, rather than by platform specific errors

Global characterization of the immune response to inoculation of aluminium hydroxide-based vaccines by RNA sequencing

xix, 195 p.En este trabajo se han analizado muestras correspondientes a un experimento de vacunación de larga duración. Múltiples ovejas fueron expuestas a varias vacunas compuestas de aluminio hidróxido como adyuvante en un periodo de 475 días, con el objetivo de estudiar el mecanismo de acción de dicho adyuvante en el sistema inmune y comprobar si es capaz de llegar a órganos distantes como el cerebro después de su inoculación. Para ello se extrajeron muestras de células mononucleares de sangre periférica y de la corteza del lóbulo parietal y se usaron para la preparación de librerías de secuenciación de ARN y microRNAs (Total RNA-seq y miRNA-seq). Las librerías se analizaron mediante herramientas bioinformáticas y se realizaron multiples análisis: 1. Expresión diferencial tanto para los datos de RNA-seq como para los de miRNA-seq; 2. Anotación de nuevos miRNAs en oveja; 3. Predicción de targets para los miRNAs y análisis de co-expresión con los datos de RNA-seq. Además, como las librerías de Total RNA-seq retienen el ARN no codificante, que esta pobremente anotado en oveja, dichos datos se usaron para la anotación de ARN circulares en oveja y se estudió si dichos ARN no-codificantes pudieran tener algún rol en la actividad del aluminio como adyuvante

Global characterization of the immune response to inoculation of aluminium hydroxide-based vaccines by RNA sequencing

xix, 195 p.En este trabajo se han analizado muestras correspondientes a un experimento de vacunación de larga duración. Múltiples ovejas fueron expuestas a varias vacunas compuestas de aluminio hidróxido como adyuvante en un periodo de 475 días, con el objetivo de estudiar el mecanismo de acción de dicho adyuvante en el sistema inmune y comprobar si es capaz de llegar a órganos distantes como el cerebro después de su inoculación. Para ello se extrajeron muestras de células mononucleares de sangre periférica y de la corteza del lóbulo parietal y se usaron para la preparación de librerías de secuenciación de ARN y microRNAs (Total RNA-seq y miRNA-seq). Las librerías se analizaron mediante herramientas bioinformáticas y se realizaron multiples análisis: 1. Expresión diferencial tanto para los datos de RNA-seq como para los de miRNA-seq; 2. Anotación de nuevos miRNAs en oveja; 3. Predicción de targets para los miRNAs y análisis de co-expresión con los datos de RNA-seq. Además, como las librerías de Total RNA-seq retienen el ARN no codificante, que esta pobremente anotado en oveja, dichos datos se usaron para la anotación de ARN circulares en oveja y se estudió si dichos ARN no-codificantes pudieran tener algún rol en la actividad del aluminio como adyuvante

Applications of Next Generation and Nanopore Sequencing for Infectious Disease Identification and Antimicrobial Resistance Detection

Infectious diseases are a major problem worldwide, in no small part due to the growing prominence of antimicrobial resistance. Current methods for species identification rely on microbiology culturing techniques and nucleic acid tests, which are time consuming, may require some a priori knowledge of infectious agents, and are limited in the information provided. To address some of these limitations, clinical diagnostics laboratories have been applying shotgun DNA sequencing for disease detection. Traditionally, metagenomic sequencing directly from clinical specimens has not been as widely used in infectious disease, due to the costs associated with producing and analyzing the data. However, sequencing is now becoming more affordable and integrated into the clinical setting. One such example is the recently released MinION sequencer from Oxford Nanopore, a portable, low-cost sequencer that connects to standard personal computers via USB. We are examining the application of the Oxford Nanopore MinION as a diagnostic aid for detecting pathogenic organisms in infectious disease, as well as acquisition of antimicrobial resistance. We hope to develop laboratory tests to identify and characterize infection causing organisms. The work for this master’s thesis focused specifically on developing and optimizing sequencing and computational techniques that can be applicable to infectious disease diagnostics. We illustrate our progress using three separate cases: 1) detection of vancomycin and carbapenem resistance in pathogens from remnant rectal swabs, 2) a clinical case study involving an extensively drug resistant strain of K. pneumoniae, and 3) long-read sequencing of clinical influenza samples. We hope to leverage the MinION’s versatility and sequence samples both from a clinical laboratory standpoint, as well as on site to locations of outbreaks

Infections in skin cancer

The increasing prevalence of skin cancer results in that it will soon equal that of all other cancers combined. Sun exposure is a well-known risk factor for its development, but despite the growing public awareness of the harmful consequences of ultraviolet radiation, the cancer incidence continues to increase, implying that other factors might also have a role in promoting this disease. Data from immunosuppressed patients reveals a 100-fold increased incidence of nonmelanoma skin carcinoma (NMSC), but an infectious etiology has not been established. However, certain human papillomaviruses (HPVs) have previously been detected in this type of cancer. We applied high throughput sequencing to different skin lesions in order to assess which organisms were present. Most viral reads (>95%) belonged to human papillomavirus. Traditionally, viral detection was performed using PCR methods. We used degenerate “general” HPV primers and multiplexed novel “specific” HPV primers in order to amplify a broad number of HPVs by PCR. This method showed a very high sensitivity, but the HPV types with low similarity to the primer sequences might have escaped amplification. Therefore, we performed an unbiased approach based on non-PCR whole genome amplification, independent of sequence information, in order to detect those “escaping” HPV types, as well as to determine if other viruses were present in the samples. Overall, we identified almost 100 putative novel HPV types in total, and characterized 4 novel HPV types (HPV 197, 200, 201 and 202). Most of the HPV types were detected in very few patients each, and at a very low viral load (below 0.5 copies/cell), except for HPV 197, which was the most commonly found virus in skin tumors (37.4% of skin lesions). Despite the higher sensitivity of PCR methods, the unbiased approach detected HPV in 37/40 condyloma acuminata that had been reported as “HPV-negative” with specific PCR techniques. Certain HPV types, including HPV 197, were not detected by PCR and only by non-PCR based methods. Therefore, more unbiased PCR-independent methods are needed to describe which organisms are most commonly present in skin lesions. The work in this thesis has expanded our knowledge of the wide genomic diversity of HPV on the skin, and finds that PCR-independent methods are needed to describe which organisms are most commonly present in skin lesions. Further studies are needed to assess any possible role of viral infections in skin cancer, elucidation of mechanistic effects and determine the direction of causality of any associations

Computational approaches for improving treatment and prevention of viral infections

The treatment of infections with HIV or HCV is challenging. Thus, novel drugs and new computational approaches that support the selection of therapies are required. This work presents methods that support therapy selection as well as methods that advance novel antiviral treatments. geno2pheno[ngs-freq] identifies drug resistance from HIV-1 or HCV samples that were subjected to next-generation sequencing by interpreting their sequences either via support vector machines or a rules-based approach. geno2pheno[coreceptor-hiv2] determines the coreceptor that is used for viral cell entry by analyzing a segment of the HIV-2 surface protein with a support vector machine. openPrimeR is capable of finding optimal combinations of primers for multiplex polymerase chain reaction by solving a set cover problem and accessing a new logistic regression model for determining amplification events arising from polymerase chain reaction. geno2pheno[ngs-freq] and geno2pheno[coreceptor-hiv2] enable the personalization of antiviral treatments and support clinical decision making. The application of openPrimeR on human immunoglobulin sequences has resulted in novel primer sets that improve the isolation of broadly neutralizing antibodies against HIV-1. The methods that were developed in this work thus constitute important contributions towards improving the prevention and treatment of viral infectious diseases.Die Behandlung von HIV- oder HCV-Infektionen ist herausfordernd. Daher werden neue Wirkstoffe, sowie neue computerbasierte Verfahren benötigt, welche die Therapie verbessern. In dieser Arbeit wurden Methoden zur Unterstützung der Therapieauswahl entwickelt, aber auch solche, welche neuartige Therapien vorantreiben. geno2pheno[ngs-freq] bestimmt, ob Resistenzen gegen Medikamente vorliegen, indem es Hochdurchsatzsequenzierungsdaten von HIV-1 oder HCV Proben mittels Support Vector Machines oder einem regelbasierten Ansatz interpretiert. geno2pheno[coreceptor-hiv2] bestimmt den HIV-2 Korezeptorgebrauch dadurch, dass es einen Abschnitt des viralen Oberflächenproteins mit einer Support Vector Machine analysiert. openPrimeR kann optimale Kombinationen von Primern für die Multiplex-Polymerasekettenreaktion finden, indem es ein Mengenüberdeckungsproblem löst und auf ein neues logistisches Regressionsmodell für die Vorhersage von Amplifizierungsereignissen zurückgreift. geno2pheno[ngs-freq] und geno2pheno[coreceptor-hiv2] ermöglichen die Personalisierung antiviraler Therapien und unterstützen die klinische Entscheidungsfindung. Durch den Einsatz von openPrimeR auf humanen Immunoglobulinsequenzen konnten Primersätze generiert werden, welche die Isolierung von breit neutralisierenden Antikörpern gegen HIV-1 verbessern. Die in dieser Arbeit entwickelten Methoden leisten somit einen wichtigen Beitrag zur Verbesserung der Prävention und Therapie viraler Infektionskrankheiten

Computational methods for single cell RNA and genome assembly resolution using genetic variation

Genetic variation and natural selection have driven the evolutionary history on this planet and are responsible for creating us and all other life as we know it. Over the past several decades, the genomic revolution has allowed us to assess population variation across humans and other species and use that to link genotypes with phenotypes and infer evolutionary histories. In this thesis, I explore computational methods for using genetic variation to demultiplex and disambiguate complex data. In single cell RNAseq, problems of batch effects, doublets, and ambient RNA are each sources of noise that impede our ability to infer the functional states of cells and compare them between experiments. One new popular new experimental design promising to solve each of these while also reducing experimental costs is mixturing multiple individuals' cells into a single experiment. In chapter 2, I present a method for clustering cells by genotype, calling doublets, and using the cross-genotype signal in singletons to estimate and remove ambient RNA. I compare this methods to other existing methods including one that requires \textit{a priori} information about the genotypes, and two which do not. I find that my method outperforms each of these methods across a wide range of data parameters and sample types. In genome assembly, the recent higher throughput and lower cost of long read sequencing has revolutionized our ability to create reference quality genomes and has revitalized the assembly community. Now, massive efforts are taking place in the Darwin Tree of Life project and the Earth Biogenome project to create reference genomes for all multicelular eukaryotic life. This will create a scientific resource for the next generation of biological science, will serve as a conservation of data that could otherwise be lost in this time of mass extinction, and will allow for a much more broad understanding of evolution and the evolutionary history of life on Earth. While much progress has been made in data quality and assembly algorithms, some problems still exist. Until recently, the DNA input requirements for long read sequencing technologies made it impossible to sequence single individuals of these species with long reads. Also, high heterozygosity makes assembly more difficult due to the inherent ambiguity between heterozygous sequence versus paralogous sequence when confronted with inexact homology. One solution to the DNA input requirements would be to pool individuals, but this only increases the heterozygosity of the sample and reduces assembly quality. In chapter 3, we present the first high quality assembly of a single mosquito using new library preparation methods with reduced DNA requirements. This reduces the number of haplotypes to two, improving the assembly quality. In chapter 4, we further address the problems brought on by heterozygosity in assembly. I present a suite of tools that use the phasing consistency of multiple heterozygous sequences as a signal for physical linkage, thus using genetic variation to our advantage rather than as a challenge to overcome. This tool creates phased, linked assemblies and phasing aware scaffolding. Further, I provide a tool for phasing aware scaffolding on existing assemblies. This includes a novel haplotype phasing algorithm with some unique beneficial properties. It is robust to non-heterozygous variants as input and can detect and correct those genotypes. And it naturally extends to polyploid genomes.Wellcome Trus

Molecular surveillance of Listeria monocytogenes in Germany to control transmission along food supply chains and to prevent human listeriosis cases

Human listeriosis is a comparatively rare but serious infectious disease. The high hospitalisation and mortality rate make it a major public health concern worldwide. Elderly and immunocompromised people as well as pregnant women are at increased risk of infection. Listeriosis is caused by consumption of food contaminated with the bacterium Listeria (L.) monocytogenes. Listeria monocytogenes is widespread in the environment and in animals. It enters the food chain either via contaminated raw animal or plant products or via cross-contamination during food processing. The overall aim of the present dissertation was to prevent cases of listeriosis in humans using state-of-the-art molecular epidemiological methods and genetic profiling of bacterial isolates. As a support to classical epidemiology, molecular typing methods are used to monitor the entry and spread of L. monocytogenes along the food chain up to the consumer. In recent years, the possibility of whole genome sequencing (WGS) has revolutionised the molecular typing of bacterial isolates. Based on the knowledge gained in the present doctoral project, the previous gold standard for fine typing, pulsed-field electrophoresis, was completely replaced by WGS in the National Reference Laboratory (NRL) for L. monocytogenes in 2018. As part of the present project, the value of WGS as a high-resolution molecular surveillance tool for L. monocytogenes has been widely communicated to stakeholders along the food chain. Based on the protocols established in the NRL during the doctoral project, the tool is now available to the national monitoring authorities. Within the dissertation project, the bioinformatic analysis of WGS data of L. monocytogenes was optimised for data exchange, a listeriosis outbreak and the spread of Listeria spp. in a food processing plant were investigated, and the significance of the European Rapid Alert System for Food and Feed (RASFF) for foodborne outbreaks was evaluated. Using WGS, contamination and infection chains can be traced with unprecedented precision and thus be stopped. However, the methods for analysing WGS data are diverse and not yet fully harmonised, which hinders data sharing. In the present work, it was shown that different WGS analysis methods for L. monocytogenes generate largely comparable results. A translation code developed within this doctoral project allows information on clusters to be exchanged between sectors (e.g. food safety, public health) and countries even without harmonised methods. This approach offers a major advantage as it allows rapid communication between stakeholders, especially in time-critical situations such as listeriosis outbreaks. An important prerequisite for the prevention of listeriosis infections is a stronger focus on industrial hygiene in food processing. This became clear in the example of a study of a poultry processing chain carried out as part of this doctoral project. By means of WGS typing, it could be shown that Listeria spp. are transferred from the animal and from the environment to the finished food product. In addition, bacteria were shown to spread to surfaces in the production environment, favouring dangerous cross-contamination. Since the introduction of WGS for typing L. monocytogenes in Germany, in cross-sectoral collaboration, a large number of listeriosis outbreaks could be clarified and stopped. This work provides insight into a large national listeriosis outbreak lasting several years, which was lead investigated in the present doctoral project. Using WGS typing, the outbreak was traced back to ready-to-eat meat products and their producer. By using forward checking as a supplement to the usual backtracking in outbreak investigations, two different clusters of listeriosis cases could be assigned to the same producer. Only through such a two-sided control strategy, combining backtracking and forward checking, can listeriosis outbreaks be significantly minimised in the future. As a result of the intensive sampling at the producer, a very diverse L. monocytogenes population was found, with some strains persisting for several years. Listeria monocytogenes was found especially in niches of the food processing environment, difficult to reach for cleaning and disinfection. This study pointed to weaknesses in industrial hygiene that resulted in the production of contaminated food and thus triggered the listeriosis outbreak. If L. monocytogenes is found in a food product, the European RASFF enables international communication of this risk and, for example, immediate product recalls. In this way, it makes an essential contribution to protecting consumers from foodborne infections. An analysis of the RASFF notifications concerning L. monocytogenes as part of this doctoral project highlighted the complexity of international processing and distribution channels. This underlines how important international cooperation is with regard to food safety. The fact that individual producers were involved in many RASFF notifications in different years again showed the influence of inadequate industrial hygiene and persistence on the spread of L. monocytogenes. In addition, the results of the study highlighted the need for food processing companies to take greater responsibility for their own operations. The success of the WGS-based surveillance strategy for L. monocytogenes, which was established as part of this doctoral project and continuously optimised over its course, was already evident during the project term. For the first time, the number of reported cases of listeriosis in Germany no longer increased in 2018, but decreased, and continued to decrease in 2019. The dissertation project has thus made a decisive contribution to controlling the spread of L. monocytogenes along the food chain and thus reducing the number of listeriosis cases in Germany.Die humane Listeriose ist eine vergleichsweise seltene, aber schwerwiegende Infektionserkrankung. Die hohe Hospitalisierungs- und Sterblichkeitsrate machen sie weltweit zu einem großen Problem für die öffentliche Gesundheit. Ältere, Schwangere und Immungeschwächte haben ein erhöhtes Infektionsrisiko. Verursacht wird die Listeriose durch den Verzehr von Lebensmitteln, die mit dem Bakterium Listeria (L.) monocytogenes kontaminiert sind. Listeria monocytogenes ist weit verbreitet in der Umwelt und Tierwelt. In die Lebensmittelkette gelangt es entweder über kontaminierte rohe tierische oder pflanzliche Produkte oder über Kreuzkontaminationen während der Verarbeitung von Lebensmitteln. Das übergeordnete Ziel der vorliegenden Arbeit war es, mittels modernster molekularepidemiologischer Methoden und genetischem Profiling von Bakterienisolaten Listeriosefälle beim Menschen zu verhindern. Neben der klassischen Epidemiologie werden molekulare Typisierungsmethoden angewendet, um den Eintrag und die Verbreitung von L. monocytogenes entlang der Lebensmittelkette bis hin zum Verbraucher aufzudecken. In den letzten Jahren wurde die molekulare Typisierung durch die Möglichkeit zur Gesamtgenomsequenzierung (WGS, whole genome sequencing) bakterieller Isolate revolutioniert. Anhand der in der vorliegenden Promotionsarbeit gewonnenen Erkenntnisse konnte der bisherige Goldstandard zur Feintypisierung, die Pulsed-Field-Gelektrophorese, im Nationalen Referenzlabor (NRL) für L. monocytogenes in 2018 komplett durch die WGS abgelöst werden. Der Wert der WGS als hochauflösendes, molekulares Überwachungstool für L. monocytogenes wurde im Rahmen des Promotionsprojekts umfassend an die Stakeholder entlang der Lebensmittelkette kommuniziert. Auf der Basis der im Zuge des Promotionsprojekts im NRL etablierten Protokolle steht das Tool den nationalen Überwachungsbehörden nun zur Verfügung. Innerhalb des Promotionsvorhabens wurde die bioinformatische Analyse von WGS-Daten von L. monocytogenes für den Datenaustausch optimiert, beispielhaft ein Listerioseausbruch und die Verbreitung von Listeria spp. in einem lebensmittelverarbeitenden Betrieb untersucht, sowie die Bedeutung des europäischen Schnellwarnsystems für lebensmittelbedingte Krankheitsausbrüche evaluiert. Mittels WGS lassen sich Kontaminations- und Infektionsketten mit beispielloser Genauigkeit nachvollziehen und so unterbrechen. Die Verfahren zur Analyse von WGS-Daten sind jedoch vielfältig und noch nicht umfassend harmonisiert, was den Datenaustausch erschwert. In der vorliegenden Arbeit wurde gezeigt, dass mit verschiedenen WGS-Analysemethoden für L. monocytogenes größtenteils vergleichbare Ergebnisse generiert werden können. Ein innerhalb der Promotionsarbeit entwickelter Übersetzungscode ermöglicht es, Informationen zu Clustern auch ohne harmonisierte Methoden zwischen Sektoren (z.B. Lebensmittelsicherheit, Öffentliches Gesundheitswesen) und Ländern auszutauschen. Dieses Vorgehen bietet einen großen Vorteil, indem es gerade in zeitkritischen Situationen wie Listerioseausbrüchen eine schnelle Kommunikation zwischen den Stakeholdern erlaubt. Eine wichtige Voraussetzung, um Listerioseinfektionen zu verhindern, ist ein stärkerer Fokus auf die Betriebshygiene in der Lebensmittelverarbeitung. Deutlich wurde dies am Beispiel einer im Rahmen der Promotionsarbeit durchgeführten Studie einer Geflügelverarbeitungskette. Mittels WGS-Typisierung konnte gezeigt werden, dass Listeria spp. vom Tier und aus der Umwelt bis in das fertige Lebensmittelprodukt übertragen werden. Zusätzlich zeigte sich eine Ausbreitung auf Oberflächen in der Produktionsumgebung, wodurch gefährliche Kreuzkontaminationen begünstigt werden. Seit der Einführung der WGS für die Typisierung von L. monocytogenes in Deutschland konnte in sektorübergreifender Zusammenarbeit eine Vielzahl von Listerioseausbrüchen aufgeklärt und beendet werden. Die vorliegende Arbeit gibt Einblick in einen großen, über mehrere Jahre anhaltenden, nationalen Listeriseausbruch, der im Rahmen des Promotionsvorhabens federführend untersucht wurde. Mittels WGS-Typisierung konnte der Ausbruch auf verzehrfertige Fleischprodukte und deren Herstellerbetrieb zurückverfolgt werden. Durch eine vorwärts gerichtete Überprüfung (forward checking) als Ergänzung zu der in Ausbruchsuntersuchungen üblichen Rückverfolgung (backtracking), konnten zwei verschiedene Cluster von Listeriosefällen demselben Betrieb zugeordnet werden. Nur durch solch eine zweiseitige Kontrollstrategie, bei der backtracking und forward checking kombiniert werden, können Listerioseausbrüche in Zukunft wesentlich minimiert werden. Als Resultat der intensiven Beprobung des Herstellerbetriebs zeigte sich eine sehr diverse L. monocytogenes-Population mit teils über mehrere Jahre persistierenden Stämmen. Listeria monocytogenes wurde insbesondere in für Reinigung und Desinfektion schwer zu erreichenden Nischen gefunden. Diese Studie deutete auf Schwachpunkte in der Betriebshygiene hin, die die Produktion kontaminierter Lebensmittel nach sich zogen und so Auslöser des Listerioseausbruchs waren. Wird L. monocytogenes in einem Lebensmittel gefunden, ermöglicht das europäische Schnellwarnsystem für Lebensmittel und Futtermittel (RASFF, Rapid Alert System for Food and Feed) eine internationale Kommunikation dieses Risikos und beispielsweise umgehende Produktrückrufe. Auf diese Weise trägt es essenziell dazu bei, Verbraucher vor lebensmittelbedingten Infektionen zu schützen. Die Analyse von RASFF-Meldungen bezüglich L. monocytogenes im Rahmen der vorliegenden Arbeit verdeutlichte die Komplexität internationaler Verarbeitungs- und Vertriebswege. Dies unterstreicht, wie wichtig auch die internationale Zusammenarbeit im Hinblick auf die Lebensmittelsicherheit ist. Die Tatsache, dass einzelne lebensmittelverarbeitende Betriebe in zahlreiche RASFF-Meldungen in unterschiedlichen Jahren involviert waren, zeigte erneut den Einfluss unzureichender Betriebshygiene und Persistenz auf die Ausbreitung von L. monocytogenes. Zudem verdeutlichten die Studienergebnisse den Bedarf einer stärkeren Eigenverantwortung lebensmittelverarbeitender Betriebe. Schon während der Laufzeit zeigte sich der Erfolg der im Rahmen des Promotionsprojekts etablierten und im Projektverlauf stetig optimierten WGS-basierten Überwachungsstrategie von L. monocytogenes. Die Zahl gemeldeter Listeriosefälle in Deutschland war in 2018 erstmals nicht mehr ansteigend, sondern rückläufig, und sank in 2019 weiter. Das Promotionsvorhaben hat damit entscheidend dazu beigetragen, die Ausbreitung von L. monocytogenes entlang der Nahrungskette zu kontrollieren und so die Zahl der Listerioseerkrankungen in Deutschland zu reduzieren