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Dynamics of Faecal Bacterial Populations in Early Infancy As Determined by Massively Parallel Sequencing

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Dynamics of Faecal Bacterial Populations in Early Infancy As Determined by Massively Parallel Sequencing Dynamics of faecal bacterial populations in early infancy as determined by massively parallel sequencing by Shantelle Claassen (CLSSHA002) SUBMITTED TO THE UNIVERSITY OF CAPE TOWN In fulfilment of the requirements for the degree MSc in Medical Microbiology March 2015 University of Cape Town Department of Clinical and Laboratory Sciences Faculty of Health Sciences University of Cape Town South Africa The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town This dissertation is lovingly dedicated in memory of my father Pieter Johannes Claassen His boundless love for family inspired my life and continues to inspire me today i Supervisors Prof. Mark. P. Nicol (MBBCh, MMed (Med Microbiol), SA FCPath (Microbiol), PhD)1,2,3 1Division of Medical Microbiology, Department of Clinical Laboratory Sciences, University of Cape Town, Cape Town, South Africa. 2Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa. 3National Health Laboratory Service of South Africa, Groote Schuur Hospital, Cape Town, South Africa. Dr. Mamadou Kaba (MD, MSc, PhD)1,2 1Division of Medical Microbiology, Department of Clinical Laboratory Sciences, University of Cape Town, Cape Town, South Africa. 2Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa. ii Declaration I, Shantelle Claassen, hereby declare that the work on which this dissertation is based on my original work (except where acknowledgements indicate otherwise) and that neither the whole work nor any part of it has been, is being, or is to be submitted for another degree in this or any other university. I empower the university to reproduce for the purpose of research either the whole or any portion of the contents in any manner whatsoever. Signature removed 18 March 2015 iii Acknowledgements I wish to express my sincere appreciation to my supervisors Prof. Mark Nicol and Dr. Mamadou Kaba whom constantly supported and encouraged me throughout my Masters degree. You have provided me with the guidance needed to excel in my own expectations and to grow as a young scientist in the field I adore most. Many thanks also to Prof. Heather Zar for providing me with the opportunity to study specimens from the Drakenstein Child Health Study. I would also like to thank everyone at the Division of Medical Microbiology for being such a warm and pleasant team to work with, I really appreciate it. Many thanks to Dr. William Nierman’s group at the J. Craig Venter Institute, United States of America, for the internship offered in Next Generation Sequencing. Special thanks to Stephanie Harris Mounaud and to Dr. Jyoti Shankar for your constant support and willingness to help during and after completing my internship. I would also like to thank Gerrit Botha at the Computational Biology Group, University of Cape Town, for your bio-informatics support. My sincerest gratitude goes to A/Prof. Sugnet Lubbe from the Department of Statistical Sciences, University of Cape Town for your undivided attention and constant support during data analysis. Thank you for your patience and willingness to help, no matter what type of analysis I suggested. I dearly appreciate the fact that you were always available and ready to answer any questions or solve any queries I had (especially after hours). Personally, I would like to honour and thank my family and friends for their unconditional love, prayers and motivation throughout the past two years. A special thanks to my mother for your constant support and love every day of my life. To my husband, Stefan Weitz, thank you for your love, support and for being my rock every day. I am grateful that my Heavenly Father bestowed His mercy, strength, wisdom and love on me. Lastly, I acknowledge the National Research Foundation (Innovation and Scarce Skills Master’s Scholarship) and the Division of Medical Microbiology, University of Cape Town for their financial assistance. iv Preface This dissertation is submitted for the degree of Master of Science in Medicine (MSc Med) in Medical Microbiology at the Division of Medical Microbiology, Department of Clinical Sciences, University of Cape Town, South Africa. The study was approved by the Human Research Ethics Committee (HREC) of the University of Cape Town, South Africa (742/2013); and supported by the Bill and Melinda Gates Foundation Global Health Grant (OPP1017641), the National Research Foundation (South Africa), the Carnegie Corporation of New York (United States of America) and the Wellcome Trust (United Kingdom). The work reported in this dissertation resulted from a collaborative effort between the Department of Paediatrics and Child Health; Department of Clinical and Laboratory Sciences, University of Cape Town, South Africa; J. Craig Venter Institute (JCVI), Maryland, United States of America; and the Department of Statistical Sciences, University of Cape Town, South Africa. The aim of this dissertation was to provide a detailed description of the experimental and computational approaches involved in generating Illumina MiSeq sequencing data for the purpose of studying the dynamics of the human stool microbiota from birth to seven months of age. The first chapter presents a detailed overview of the literature in context of the project. The second chapter mainly focusses on the experimental and computational approaches used to generate Illumina MiSeq sequencing data from faecal specimens. Work reported in the second chapter resulted from the following collaborative efforts. The MSc candidate performed nucleic acid extraction sections at the Division of Medical Microbiology, University of Cape Town, based on the published work by Claassen et al. (2013) in the Journal of Microbiological Methods. The MSc candidate prepared the DNA library for sequencing under guidance of staff from Dr. William Nierman’s group at JCVI. JCVI’s sequencing team performed the Illumina Miseq run. Dr. Jyoti Shankar from Dr. William Nierman’s laboratory performed the bioinformatics workflow to correct for sequencing artifacts, and to construct and classify operational taxonomic units. Further quality checks (such as contamination and reproducibility assessment) were designed by the MSc candidate and A/Prof. Sugnet Lubbe from the Department of Statistical Sciences, University of Cape Town. The final chapter of this dissertation focusses on infant faecal bacterial dynamics from birth up until seven months of life. This chapter uses the quality controlled data generated from Chapter 2 to study the infant faecal microbiota from maternal and faecal specimens sampled at birth as well as a subset of infants sampled up until seven months of age. The data analysis system for Chapter 3 was designed by the MSc candidate and A/Prof. Sugnet Lubbe from the Department of Statistical Sciences, University of Cape Town. The submitted material is the work of the MSc candidate, unless stated otherwise by acknowledgments. v Table of content Abbreviations and acronyms ...................................................................................................... 1 Glossary....................................................................................................................................... 2 References .................................................................................................................................................... 4 ABSTRACT........................................................................................................................... 5 GENERAL INTRODUCTION ....................................................................................................... 6 References .................................................................................................................................................... 7 CHAPTER 1 ......................................................................................................................... 9 THE HUMAN GASTROINTESTINAL TRACT MICROBIOME EARLY IN LIFE - A LITERATURE REVIEW ................ 9 Summary ..................................................................................................................................................... 10 1.1 The human gastrointestinal microbiota is a vital ‘organ’ ..................................................................... 11 1.1.1 The epithelial barrier function of the GIT microbiota .......................................................................... 11 1.1.2 Colonization resistance against enteric pathogens ............................................................................. 12 1.1.3 Maturation of the host’s immune system is modulated by GIT microbiota ........................................ 12 1.1.3.1 Immune-related effects of the GIT microbiota in preventing bacterial infection ....................... 12 1.1.3.2 Complex bacterial profiles in the GIT also shape systemic immunity. ........................................ 15 1.1.4 Metabolic functions of GIT microbiota ................................................................................................ 17 1.2 How to study our microbial inhabitants? ...........................................................................................
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