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Molecular Genotyping and Whole Genome Sequencing of Canadian Cyclospora Cayetanensis Specimens

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Molecular Genotyping and Whole Genome Sequencing of Canadian Cyclospora Cayetanensis Specimens Molecular Genotyping and Whole Genome Sequencing of Canadian Cyclospora cayetanensis Specimens by Christine Allison Yanta A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Pathobiology Guelph, Ontario, Canada © Christine Yanta, May, 2021 ii ABSTRACT MOLECULAR GENOTYPING AND WHOLE GENOME SEQUENCING OF CANADIAN CYCLOSPORA CAYETANENSIS SPECIMENS Christine Yanta Advisors: University of Guelph, 2021 Professor John R. Barta Dr. Rebecca A. Guy To improve our understanding of the genetic diversity of the human-infecting coccidium, Cyclospora cayetanensis, 160 clinical fecal samples representing Canadian cyclosporiasis cases, from four provinces (ON=119, QC=24, BC=7, NL=10) identified between 2010 and 2020, were genotyped using a next generation sequencing targeted amplicon approach consisting of eight markers. Genotyping data were collected for at least one marker for 96.2% (154/160) of specimens and only 36.9% (59/160) of specimens had genotyping data for all eight markers. We identified eighteen genetic clusters from the 79.4% (127/160) of specimens that successfully clustered. Whole genome sequence assemblies were generated for five of the cyclosporiasis cases that were genotyped, including a hybrid assembly that improved the current reference assembly (GCF_002999335.1); this refined assembly was 44.2 Mbp in length (297 contigs, N50 value of 654019). These first molecular data generated from Canadian cyclosporiasis cases will support and inform future epidemiological investigations aimed at mitigating cyclosporiasis outbreaks. iii ACKNOWLEDGEMENTS The success of this Master’s research project would have not been possible without the help and support of the amazing people around me. First and foremost, I would like to thank my co-advisor Dr. Rebecca Guy. Dr. Guy first welcomed me as a co-op student in her parasitology lab and has since provided me numerous opportunities to learn new and fascinating parasite and genomics-related things. Without her full confidence and continuous support, I would have never flourished to become the researcher and person I am today. I am also very grateful for my other co-advisor Professor John Barta. Throughout my research, he always believed in my abilities and provided support when needed. I would also like to thank my advisory committee member, Dr. Claire Jardine, for taking the time to provide feedback and advice throughout the project. I would also like to thank my family at NML Guelph. You welcomed me with open arms and since then were always willing to help a girl out. I would especially like to thank the parasitology group, Laura Martin and Marisa Rankin, for the endless support and making the days stuck in the biological safety cabinet with clinical specimens fly by. Thank you to Amy Feddema for being one of my biggest cheerleaders and always having the best coffee break chats. Last but certainly not least, I would like to give a huge shout-out to my family for all of their unconditional love and emotional support throughout this journey. To my parents, Donald and Allice, words simply cannot express how grateful I am to have the both of you in my life. Without your encouragement to chase my dreams and discover my potential, I would not be where I am and who I am today. I thank my brother Jeff for always being there for me and helping me anyway that you could. Finally, I want to thank my partner, Tim Hill, for being the rock in my life. Thank you for always listening, supporting, feeding, and believing in me through the thick and thin of times. You showed me how to simply take a break when things get tough and to enjoy the fresh- air and nature around us. I am so fortunate to have met you and cannot wait to see what is next to come in our journey together. iv TABLE OF CONTENTS ABSTRACT ................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ......................................................................................................................... vi LIST OF FIGURES ...................................................................................................................... vii LIST OF ABBREVIATIONS ...................................................................................................... viii LIST OF APPENDICES ................................................................................................................ ix DECLARATION OF WORK PERFORMED ................................................................................ x CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW ............................................... 1 1.0 Introduction ........................................................................................................................... 1 2.0 Biological Characteristics ..................................................................................................... 3 2.1 Morphology....................................................................................................................... 3 2.2 Life Cycle.......................................................................................................................... 4 2.3 Infectivity .......................................................................................................................... 5 3.0 Epidemiology ........................................................................................................................ 6 3.1 Geographic Distribution and Prevalence .......................................................................... 6 3.2 Transmission ..................................................................................................................... 6 3.3 Risk Factors and Symptoms.............................................................................................. 9 3.4 Treatment .......................................................................................................................... 9 4.0 Detection ............................................................................................................................... 9 4.1 Clinical Detection Methods .............................................................................................. 9 4.2 Detection in Produce ....................................................................................................... 11 5.0 Molecular Characteristics ................................................................................................... 13 5.1 Molecular Structure ........................................................................................................ 13 5.2 Gene Targets and Schemes ............................................................................................. 17 6.0 Conclusion .......................................................................................................................... 22 STUDY OBJECTIVES AND RATIONALE ............................................................................... 23 CHAPTER 2: EVALUATION OF THE ENSEMBLE-BASED MLST SCHEME ..................... 25 2.1 Abstract ............................................................................................................................... 25 2.2 Introduction ......................................................................................................................... 25 2.3 Materials and Methods ........................................................................................................ 27 2.3.1 Clinical Fecal Specimens ............................................................................................. 27 2.3.2 Ethics............................................................................................................................ 28 2.3.3 DNA Extraction ........................................................................................................... 28 2.3.4 PCR Amplification and Amplicon Clean-Up .............................................................. 28 2.3.5 DNA Sequencing ......................................................................................................... 29 2.3.6 Sequence Analysis and Clustering ............................................................................... 29 2.4 Results ................................................................................................................................. 32 2.4.1 Sequencing Success ..................................................................................................... 32 2.4.2 Clustering ..................................................................................................................... 39 2.5 Discussion ........................................................................................................................... 41 2.6 Conclusion .......................................................................................................................... 44 v CHAPTER 3: WHOLE GENOME SEQUENCING OF CANADIAN CYCLOSPORA CAYETANENSIS SPECIMENS ............................................................................... 45 3.1 Abstract ..............................................................................................................................
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