UNIVERSITY OF OKLAHOMA GRADUATE COLLEGE STUDYING MICROBIAL COMMUNITY DIVERSITIES BY DEVELOPING HIGH- THROUGHPUT EXPERIMENTAL TECHNIQUES AND COMPUTATIONAL TOOLS A DISSERTATION SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY By YUJIA QIN Norman, Oklahoma 2018 STUDYING MICROBIAL COMMUNITY DIVERSITIES USING HIGH-THROUGHPUT TECHNIQUES AND COMPUTATIONAL TOOLS A DISSERTATION APPROVED FOR THE DEPARTMENT OF MICROBIOLOGY AND PLANT BIOLOGY BY Dr. Jizhong Zhou, Chair Dr. Meijun Zhu Dr. Michael J. McInerney Dr. Henry Neeman Dr. Bradley S. Stevenson © Copyright by YUJIA QIN 2018 All Rights Reserved. Acknowledgements At the end of the journey pursuing my doctoral degree in Microbiology at University of Oklahoma, I am deeply grateful for many people who have helped and accompanied me during these years, without whom, I would never get this far. First, my advisor, Jizhong Zhou, who is an intelligent and incredibly hard-working scientist. Besides the financial support over the years, Dr. Zhou has provided me guidance, encouragement, and much patience. He is the person who led me into the field of environmental microbiology, and I really appreciated the vast training I received from the projects I’ve got involved in and the freedom he provided while exploring the field of interest. Dr. Ye Deng, who played a role as my second advisor, is also the person I would like to thank the most. Ye provide many helps and supports during his year working with me. I learned a lot from him, such as the data analysis methods and scientific writing skills, which would also benefit me for life. I owe great thanks to my committee members: Dr. Meijun Zhu, Dr. Henry Neeman, Dr. Michael McInerney, and Dr. Bradley Steven for serving as my committee members for this long time. As a graduate student trying to develop her own background in a multidisciplinary area, I deeply appreciated their guidance throughout this whole degree period, by providing valuable suggestions for every step in pursuing this degree. Many thanks to the people for their help during my research, especially Dr. Liyou Wu, Dr. Joy Van Nostrand, Dr. Daliang Ning, Dr. Qichao Tu, who provided their kind help and cooperation in the projects I’ve been working on. And Missy Lee who served as a secretary in the lab and makes everything else smooth and comfortable. My final sincerest gratitude belongs to my family, for their continuous encouragements and support in my whole life. It is them who helped me to survive all the iv stress for years and not letting me give up. My friends, roommates, a journey without them would have been not so worth memorizing. v Table of Contents Acknowledgements ......................................................................................................... iv Table of Contents ............................................................................................................ vi List of Tables ................................................................................................................. viii List of Supplementary Tables .......................................................................................... ix List of Figures ................................................................................................................... x List of Supplementary Figures ....................................................................................... xii Abstract .......................................................................................................................... xiii Chapter 1: Introduction ..................................................................................................... 1 1.1 Microbial biodiversity and current challenges ............................................... 1 1.2 Amplicon sequencing technology and taxonomic diversities ........................ 3 1.3 Functional diversities and functional gene array (GeoChip) .......................... 5 1.3.1 Measuring functional diversity ................................................................. 5 1.3.2 Microbial functional diversity and GeoChip ............................................ 9 1.4 Foci of this study .......................................................................................... 11 Chapter 2: Phasing amplicon sequencing on Illumina Miseq ........................................ 15 2.1 Abstract ......................................................................................................... 15 2.2 Introduction .................................................................................................. 16 2.3 Material and Methods ................................................................................... 19 2.3.1 Samples, mock community design and DNA extraction ........................ 19 2.3.2 PCR primers and amplification .............................................................. 21 2.3.3 Illumina MiSeq sequencing .................................................................... 24 2.3.4 Data analysis and amplicon sequence data analysis pipeline ................. 26 2.4 Results .......................................................................................................... 30 2.4.1 Basic sequencing properties using phasing strategy .............................. 30 2.4.2 Effective reads number and read lengths ................................................ 32 2.4.3 Error rate analysis using mock communities .......................................... 34 2.4.4 Potential bias source for OTU composition in mock communities ........ 37 2.5 Discussion ..................................................................................................... 39 2.6 Conclusion .................................................................................................... 41 Chapter 3: The diversity pattern of soil fungal microbial community in North America forest systems ..................................................................................................... 44 3.1 Abstract ......................................................................................................... 44 3.2 Introduction .................................................................................................. 46 3.3 Material and Methods ................................................................................... 48 3.3.1 Six forest sites and sampling strategy .................................................... 48 3.3.2 Metadata collection ................................................................................ 49 3.3.3 DNA extraction and Illumina sequencing .............................................. 50 3.3.4 Sequence processing and annotation ...................................................... 52 3.3.5 Statistical methods .................................................................................. 54 3.4 Results .......................................................................................................... 57 3.4.1 Sequencing results .................................................................................. 57 3.4.2 Fungal community composition across the six forest sites .................... 58 3.4.3 α-diversity pattern and its drivers ........................................................... 61 3.4.4 β-diversity and distance-decay pattern ................................................... 65 vi 3.5 Discussion ..................................................................................................... 68 3.6 Conclusions ........................................................................................................ 71 Chapter 4: Microbial Functional diversity and Ecosystem functioning ......................... 73 Abstract ......................................................................................................... 73 Introduction .................................................................................................. 75 Mathematical framework of functional diversity ......................................... 76 4.3.1 Functional traits and GeoChip database ................................................. 76 4.3.2 Rao’s quadratic entropy .......................................................................... 77 4.3.2.1 Functional diversity ................................................................................ 78 4.3.2.2 Partition of functional diverity (α, β and γ-diversity) ............................. 79 4.3.2.3 Corrected functional α, β and γ diversity ............................................... 81 4.3.2.4 Functional redundancy ........................................................................... 81 4.3.2.5 Community level functional diversity and redundancy .......................... 83 4.3.3 Quantifying distances between taxa ....................................................... 83 4.3.4 Pipeline construction .............................................................................. 88 Applications and results ............................................................................... 91 4.4.1 Groundwater dataset ............................................................................... 91 4.4.2 Linking functional diversity to ecosystem functions .............................. 93 4.4.3 Shifts of the overall functional structures of microbial communities .... 99 Discussion ................................................................................................... 102 Conclusion