Comparative genomics of pathogenic lineages of Listeria monocytogenes - A bioinformatic approach Inaugural-Dissertation (Cumulative thesis) Submitted to the Faculty of Medicine in fulfilment of the requirements for the degree of Dr. biol. hom. of the Faculty of Medicine of the Justus-Liebig-University Gießen By Carsten Tobias Künne of Westerburg Gießen 2012 From the Institute of Medical Microbiology Director: Prof. Dr. Trinad Chakraborty Universitätsklinikum Gießen und Marburg GmbH Standort Gießen First supervisor and Committee Member: Prof. Dr. Trinad Chakraborty Committee Member: Prof. Dr. Klaus T. Preissner Date of Doctoral Defense: 18.06.2013 Table of Contents 1 LIST OF PUBLICATIONS ........................................................................................... 1 1.1 PUBLICATION I - COMPLETE GENOME SEQUENCE OF LISTERIA SEELIGERI , A NONPATHOGENIC MEMBER OF THE GENUS LISTERIA ................................................. 1 1.2 PUBLICATION II - COMPARATIVE ANALYSIS OF PLASMIDS IN THE GENUS LISTERIA ...................................................................................................................... 1 1.3 PUBLICATION III - GENOME -WIDE IDENTIFICATION OF SMALL RNA S IN THE OPPORTUNISTIC PATHOGEN ENTEROCOCCUS FAECALIS ............................................. 1 1.4 PUBLICATION IV - COMPLETE SEQUENCES OF PLASMIDS FROM THE HEMOLYTIC -UREMIC SYNDROME -ASSOCIATED ESCHERICHIA COLI STRAIN HUSEC41 ................................................................................................................... 2 1.5 PUBLICATION V - SRNA DB : A SMALL NON -CODING RNA DATABASE FOR GRAM -POSITIVE BACTERIA ......................................................................................... 2 1.6 PUBLICATION VI - COMPARATIVE GENOMICS AND TRANSCRIPTOMICS OF LINEAGES I, II, AND III STRAINS OF LISTERIA MONOCYTOGENES............................... 2 1.7 PUBLICATION VII – DYNAMIC INTEGRATION HOTSPOTS AND MOBILE GENETIC ELEMENTS SHAPE THE GENOME STRUCTURE OF THE SPECIES LISTERIA MONOCYTOGENES ........................................................................................................ 2 2 INTRODUCTION .......................................................................................................... 3 2.1 FROM SANGER TO NEXT -GENERATION SEQUENCING (NGS) ................................... 3 2.2 FROM READS TO REPLICONS - ASSEMBLY AND FINISHING STRATEGIES ................... 6 2.3 A PRIMER FOR SEQUENCE COMPARISON .................................................................... 8 2.4 SMALL NON -CODING REGULATORY RNA IDENTIFICATION ...................................... 9 2.5 CAPTURING DIVERSITY – GENOME , TRANSCRIPTOME , PAN -GENOME , META - GENOME ..................................................................................................................... 10 2.6 ANNOTATION ............................................................................................................. 12 2.7 PHYLOGENETIC TREES ............................................................................................. 13 2.8 COMPARATIVE VISUALIZATION ................................................................................ 15 2.9 GENUS LISTERIA ........................................................................................................ 16 I 2.10 SPECIES L. MONOCYTOGENES .................................................................................... 16 2.11 RESISTANCE TO BACTERIOPHAGES .......................................................................... 17 2.12 MAIN OBJECTIVES OF THIS STUDY ........................................................................... 18 3 RESULTS AND DISCUSSION ................................................................................... 19 3.1 BIOINFORMATIC PIPELINES ...................................................................................... 19 3.1.1 ASSEMBLY AND SCAFFOLDING OF 454 PYROSEQUENCING DATA .......................... 19 3.1.2 FINDING SPECIFIC PRIMERS FOR THE FINISHING PHASE OF GENOME SEQUENCING ........................................................................................................ 20 3.1.3 JOINING CONTIGS AND GAP CLOSURE PCR S ......................................................... 21 3.1.4 COMPARATIVE ANALYSES USING GECO ............................................................. 23 3.1.5 ANNOTATION PIPELINE ........................................................................................ 25 3.1.6 TOTAL REPLICON VISUALIZATIONS USING MAUVE AND GENOME VIZ IDENTIFY GLOBAL SIMILARITIES .......................................................................................... 26 3.1.7 CREATING AND ASSESSING PHYLOGENIES ............................................................ 29 3.1.8 PAN -GENOME DISTRIBUTION AND PREDICTION OF TOTAL SIZE ............................. 32 3.1.9 DELINEATING THE IMPACT OF DUPLICATION AND HORIZONTAL GENE TRANSFER ............................................................................................................ 33 3.1.10 COMPARATIVE IDENTIFICATION OF HYPERDYNAMIC HOTSPOTS AND MOBILE GENETIC ELEMENTS .............................................................................................. 34 3.1.11 FINDING AND DESCRIBING GENES WHICH SUPPORT TAXONOMIC OR PHENOTYPICAL DIVISIONS .................................................................................... 36 3.1.12 BIOINFORMATIC IDENTIFICATION OF SMALL NON -CODING REGULATORY RNA S .................................................................................................................. 37 3.1.13 IDENTIFICATION AND VISUALIZATION OF CRISPR ADAPTIVE IMMUNE SYSTEMS .............................................................................................................. 38 3.1.14 PROBLEMS ENCOUNTERED AND POSSIBLE SOLUTIONS TO FURTHER STREAMLINE ANALYSES ....................................................................................... 41 3.2 SUMMARIES OF PUBLICATIONS ................................................................................ 42 3.2.1 COMPLETE GENOME SEQUENCE OF LISTERIA SEELIGERI , A NON -PATHOGENIC MEMBER OF THE GENUS LISTERIA ......................................................................... 42 3.2.1.1 Methods and Contribution ............................................................................... 42 II 3.2.1.2 Summary ......................................................................................................... 42 3.2.2 COMPARATIVE ANALYSIS OF PLASMIDS IN THE GENUS LISTERIA ........................... 42 3.2.2.1 Methods and Contribution ............................................................................... 42 3.2.2.2 Summary ......................................................................................................... 43 3.2.3 GENOME -WIDE IDENTIFICATION OF SMALL RNA S IN THE OPPORTUNISTIC PATHOGEN ENTEROCOCCUS FAECALIS ................................................................... 44 3.2.3.1 Methods and Contribution ............................................................................... 44 3.2.3.2 Summary ......................................................................................................... 44 3.2.4 COMPLETE SEQUENCES OF PLASMIDS FROM THE HEMOLYTIC -UREMIC SYNDROME -ASSOCIATED ESCHERICHIA COLI STRAIN HUSEC41 .......................... 46 3.2.4.1 Methods and Contribution ............................................................................... 46 3.2.4.2 Summary ......................................................................................................... 46 3.2.5 SRNA DB : A SMALL NON -CODING RNA DATABASE FOR GRAM -POSITIVE BACTERIA ............................................................................................................. 46 3.2.5.1 Methods and Contribution ............................................................................... 46 3.2.5.2 Summary ......................................................................................................... 46 3.2.6 COMPARATIVE GENOMICS AND TRANSCRIPTOMICS OF LINEAGES I, II, AND III STRAINS OF LISTERIA MONOCYTOGENES ................................................................ 47 3.2.6.1 Methods and Contribution ............................................................................... 47 3.2.6.2 Summary ......................................................................................................... 47 3.2.7 DYNAMIC INTEGRATION HOTSPOTS AND MOBILE GENETIC ELEMENTS SHAPE THE GENOME STRUCTURE OF THE SPECIES LISTERIA MONOCYTOGENES .................. 48 3.2.7.1 Methods and Contribution ............................................................................... 49 3.2.7.2 Summary ......................................................................................................... 49 4 SUMMARY ................................................................................................................... 51 5 ZUSAMMENFASSUNG ............................................................................................. 53 6 ABBREVIATIONS ...................................................................................................... 56 7 REFERENCES ............................................................................................................. 58 8 ERKLÄRUNG ZUR DISSERTATION ..................................................................... 66 III 9 ACKNOWLEDGEMENTS ........................................................................................