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Dynamics of the Gut Virome and Bacterial Microbiome in Pediatric Crohn’S Disease Patients

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Dynamics of the Gut Virome and Bacterial Microbiome in Pediatric Crohn’S Disease Patients DYNAMICS OF THE GUT VIROME AND BACTERIAL MICROBIOME IN PEDIATRIC CROHN’S DISEASE PATIENTS By Casey Michael Arthur Jones Submitted in partial fulfilment of the requirements for the degree of Master of Science at Dalhousie University Halifax, Nova Scotia December 2018 © Copyright by Casey Michael Arthur Jones, 2018 Dedication To my family, friends, coaches, teammates, and mentors, who have been there through it all. ii TABLE OF CONTENTS LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii ABSTRACT ....................................................................................................................... ix LIST OF ABBREVIATIONS USED ................................................................................. x ACKNOWLEDGEMENTS .............................................................................................. xii CHAPTER 1: INTRODUCTION ................................................................................. 1 1.1 CROHN’S DISEASE .......................................................................................... 1 1.1.1 IBD EPIDEMIOLOGY .............................................................................. 1 1.1.2 DIAGNOSIS OF IBD ................................................................................. 2 1.1.3 PATHOGENESIS OF CROHN’S DISEASE ............................................. 3 1.1.3.1 GENETICS OF IBD ..................................................................................... 3 1.1.3.2 IMMUNOLOGY .......................................................................................... 5 1.1.3.3 ENVIRONMENT ......................................................................................... 6 1.1.4 TREATMENTS FOR CROHN’S DISEASE ............................................. 7 1.1.4.1 PHARMACOLOGICAL THERAPIES ...................................................... 8 1.1.4.2 EXCLUSIVE ENTERAL NUTRITION (EEN) IN TREATMENT FOR IBD ............................................................................................................ 11 1.2 THE GUT MICROBIOME ............................................................................... 13 1.3 THE GUT VIROME ......................................................................................... 17 1.4 ANALYSING THE MICROBIOME ................................................................ 19 1.4.1 DNA SEQUENCING ............................................................................... 19 1.4.2 BIOINFORMATIC APPROACHES ........................................................ 20 1.5 THE MICROBIOME IN IBD ........................................................................... 22 iii 1.6 OBJECTIVES ................................................................................................... 25 CHAPTER 2: MATERIALS AND METHODS ....................................................... 26 2.1 MAREEN STUDY RECRUITMENT .............................................................. 26 2.2 VIRUS LIKE PARTICLE ENRICHMENT PROTOCOL ................................ 26 2.3 LIBRARY PREPARATION AND SEQUENCING ......................................... 27 2.4 VIRAL SEQUENCE PROCESSING AND ANALYSIS ................................. 27 2.5 16S WETLAB PROTOCOL ............................................................................. 29 2.6 16S RIBOSOMAL RNA SEQUENCING ANNOTATION ............................. 29 2.7 STATISTICAL METHODS ............................................................................. 30 2.8 MACHINE LEARNING ................................................................................... 31 CHAPTER 3: THE GUT VIROME.................................................................................. 33 3.1 VALIDATION OF VLP PREPARATION ....................................................... 34 3.2 COMPARISON OF BIOINFORMATIC METHODS FOR VIRAL ANNOTATION ................................................................................................ 37 3.3 REFERENCE-INDEPENDENT APPROACHES ARE UNINFORMATIVE FOR CLASSIFYING THE GUT VIROME ...................................................... 39 3.4 REFERENCE SEARCHES PRODUCE VALUABLE INSIGHT ON THE PEDIATRIC CD GUT VIROME ...................................................................... 42 3.5 MACHINE LEARNING IDENTIFIES SUBTLE CHANGES IN THE GUT VIROME RELATED TO CLINICAL OUTCOMES ....................................... 50 3.6 SUMMARY ...................................................................................................... 51 CHAPTER 4: THE BACTERIOME OF PEDIATRIC CD .............................................. 52 4.1 ALPHA DIVERSITY OF THE BACTERIOME DIFFERS WITH FECAL CALPROTECTIN LEVELS ............................................................................. 52 4.2 BACTERIAL TAXA DIFFER WITH FECAL CALPROTECTIN LEVEL AND REMISSION STATUS ..................................................................................... 54 iv 4.3 BACTERIAL TAXA SIGNIFICANTLY MODEL FCP LEVELS .................. 56 4.4 SUMMARY ...................................................................................................... 58 CHAPTER 5: VIROME-BACTERIOME INTERACTIONS .......................................... 59 5.1 HIGH LEVEL BACTERIA – VIROME ASSOCIATIONS ............................. 59 5.2 KNOWN PHAGE-BACTERIA INTERACTIONS DO NOT CORRELATE WELL IN PEDIATRIC CD PATIENTS ........................................................... 62 5.3 ENTEROCOCCUS PHAGE IS ASSOCIATED WITH ITS HOST AND DISEASE ACTIVITY IN ONE PATIENT ....................................................... 62 5.4 SUMMARY ...................................................................................................... 64 CHAPTER 6: DISCUSSION ............................................................................................ 65 6.1 VIRUS LIKE PARTICLE PREPARATIONS .................................................. 65 6.2 REFERENCE BASED BIOINFORMATICS ARE OPTIMAL FOR ANNOTATION OF THE GUT VIROME ........................................................ 68 6.3 VIRAL DIVERSITY IS ASSOCIATED WITH SEVERAL CLINICAL CHARACTERISTICS ...................................................................................... 69 6.4 BACTERIOME OF CD .................................................................................... 72 6.5 THE GUT VIROME AND BACTERIOME DISPLAY MODEST INTERACTIONS IN THE PEDIATRIC CD GUT ........................................... 74 6.6 LIMITATIONS AND FUTURE DIRECTIONS .............................................. 76 6.7 CONCLUSIONS............................................................................................... 78 REFERENCES ................................................................................................................. 79 v LIST OF TABLES Table 1 MAREEN Study cohort information .............................................................. 33 Table 2 Shannon diversity is correlated with selected clinical phenotypes ............... 45 Table 3 Top 20 viral species important for regression of FCP levels ......................... 50 Table 4 Differentially abundant bacterial taxa between samples with high and low fecal calprotectin samples ............................................................................................... 55 Table 5 Top 20 bacterial taxa important for regression of FCP levels ...................... 57 vi LIST OF FIGURES Figure 1 Virally-enriched samples show a greater percent read mapping to viral protein database .............................................................................................................. 35 Figure 2 Stacked bar chart depicting relative abundance of top 10 most abundant phages in a blank run sample ........................................................................................ 36 Figure 3 Stacked bar chart depicting relative abundance of 10 most abundant viruses in Modulen EEN formula .................................................................................. 37 Figure 4 Performance of selected bioinformatic tools of annotating viral metagenomic sequencing reads in virally enriched samples ....................................... 38 Figure 5. Shannon Diversity of each MAREEN patient across all timepoints .......... 39 Figure 6 Viral genome bin diversity does not differ between samples with low FCP and high FCP levels ........................................................................................................ 40 Figure 7 Viral genome bin dissimilarity in all MAREEN patients ............................ 41 Figure 8 Boxplot of most abundant viral families observed in MAREEN patients.. 43 Figure 9 Viral species diversity is higher in samples with low disease activity compared to samples with high disease activity ........................................................... 44 Figure 10 Stacked bar charts of viral families grouped by inflammation level and time sampled .................................................................................................................... 46 Figure 11 Patients that undergo sustained remission after EEN administration do not have distinct gut virome compositions.................................................................... 47 Figure 12 The gut virome significantly varies by patient ............................................ 48 Figure
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