Taxonomic and Environmental Annotation of Bacterial 16S rRNA gene sequences via Shannon Entropy and Database Metadata Terms by Ali Zeeshan Ijaz Hawkesbury Institute for the Environment Western Sydney University, Australia Thesis submitted: February 2017 A thesis submitted for fulfilment of the requirements of the degree of Doctor of Philosophy Dedication I would like to dedicate my thesis to my beloved grandfather, Iqbal Ahmad and my elder sister Sidrah Ijaz, who have taught me to work hard for the things that I aspire to achieve. Thank you for your support along the way. Acknowledgements I am greatly indebted to my PhD supervisor, Prof. Brajesh Singh and co-supervisor Dr. Christopher Quince for identifying and streamlining the research to be undertaken for the completion of my PhD at Hawkesbury Institute for the Environment, Western Sydney University. Their professional competence, supervision and continued support in all areas of personal and professional interest have been paramount towards completion of my studies with zeal and enthusiasm. All along I have had the opportunity to interact and enjoy the able guidance and support of other professional and competent people, with the foremost being Dr. Thomas Jeffries, to whom I extend my special thanks for his unending patience and time devotion to keep me on track and encouragement that made it possible to complete my studies. His competence and grasp of knowledge facilitated an environment that made it possible to perform my research in the right direction. I would also like to thank Jasmine Grinyer for her support in various aspects of paperwork. Lastly, I would like to thank my family for being extremely understanding and supportive all along the study period, including my brother Dr. Umer Zeeshan Ijaz, a Research fellow at University of Glasgow. Their support and encouragement gave me the resolve to complete my PhD while being under a serious ailment. Statement of Authentication The work presented in this thesis is, to the best of my knowledge and belief, original except as acknowledged in the text. It contains no material previously published or written by another person. I hereby declare that I have not submitted this material, either in full or in part, for a degree at this or any other institute. Signed Ali Zeeshan Ijaz 1st June 2018 Table of Contents Table of Contents ..................................................................................................................................... i List of Tables ........................................................................................................................................... iii List of Figures ........................................................................................................................................ vii List of Abbreviations ........................................................................................................................... xi Abstract..................................................................................................................................................... xii Chapter 1: General Introduction ................................................................................................... 1 1.1 Importance of Microbial Community Analysis ....................................................... 1 1.2 Taxonomic Annotation using Conserved Marker Genes .................................... 2 1.3 Environmental Annotation of Sequences ................................................................ 16 1.4 Knowledge Gaps................................................................................................................... 19 1.5 Aims and Objectives ........................................................................................................... 22 Chapter 2: Exploiting The Evolutionary Conservation of 16S rRNA Gene via Shannon Entropy ................................................................................................................................. 25 2.1 Introduction ........................................................................................................................... 25 2.2 Materials and Methods ..................................................................................................... 30 2.3 Results ...................................................................................................................................... 52 2.4 Discussion ............................................................................................................................... 60 2.5 Conclusion .............................................................................................................................. 64 Chapter 3: TaxaSE: Taxonomic Annotation via Shannon Entropy .............................. 66 3.1 Introduction ........................................................................................................................... 66 3.2 Materials and Methods ..................................................................................................... 72 3.3 Results ...................................................................................................................................... 82 3.4 Discussion ............................................................................................................................ 104 i 3.5 Conclusion ........................................................................................................................... 108 Chapter 4: Assigning Environmental Terms to Sequences using SEQenv ........... 110 4.1 Introduction ........................................................................................................................ 110 4.2 Materials and Methods .................................................................................................. 115 4.3 Results ................................................................................................................................... 127 4.4 Discussion ............................................................................................................................ 144 4.5 Conclusion ........................................................................................................................... 152 Chapter 5: Final Conclusion and Future Work .................................................................. 154 5.1 Conclusion ........................................................................................................................... 154 5.2 Future work ........................................................................................................................ 157 Appendix A. ......................................................................................................................................... 159 References ............................................................................................................................................ 189 ii List of Tables Table 2-1: Database as a m x n matrix where rows are sequences and columns represent alignment positions ............................................................................................ 32 Table 2-2: List of thresholds between 0 and 1 used for calculation of precision and recall ........................................................................................................................................ 42 Table 2-3: List of taxa removed at genus, family and class level from SILVA database.......................................................................................................................................... 45 Table 2-4: List of tools and scripts............................................................................................... 48 Table 2-5: Area under the curve for whole SILVA dataset based validation for both the percentage identity and Shannon entropy approach ............................ 53 Table 2-6: Area under the curve for removal of genera based validation for both percentage identity and Shannon entropy approach ............................................... 54 Table 2-7: Area under the curve for removal of families based validation for both percentage identity and Shannon entropy approach ............................................... 56 Table 2-8: Area under the curve for removal of class based validation for both percentage identity and Shannon entropy approach ............................................... 57 Table 3-1: Lists of tools and scripts developed for the TaxaSE pipeline. .................. 73 Table 3-2: Sample data used for real amplicon dataset analysis .................................. 75 Table 3-3: Thresholds selected for the TaxaSE system at different taxa levels ..... 79 Table 3-4: ADONIS results for OTU comparison at 97% similarity between TaxaSE and QIIME ..................................................................................................................... 89 Table 3-5: ANOSIM results for OTU comparison at 97% similarity between TaxaSE and QIIME ..................................................................................................................... 90 iii Table 3-6: ADONIS results for distinct taxonomic annotation comparison between TaxaSE, QIIME at 99% OTU similarity and QIIME at 97% OTU similarity ..................................................................................................................................... 102