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Optimized Analysis of the Lung Allograft Microbiota from Bronchoalveolar Lavage Fluid

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Optimized Analysis of the Lung Allograft Microbiota from Bronchoalveolar Lavage Fluid Optimized Analysis of the Lung Allograft Microbiota from Bronchoalveolar Lavage Fluid by Janice Evana Prescod A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Laboratory of Medicine and Pathobiology University of Toronto © Copyright by Janice Evana Prescod 2018 Optimized Analysis of the Lung Allograft Microbiota from Bronchoalveolar Lavage Fluid Janice Prescod Master of Science Department of Laboratory of Medicine and Pathobiology University of Toronto 2018 Abstract Introduction: Use of bronchoalveolar lavage fluid (BALF) for analysis of the allograft microbiota in lung transplant recipients (LTR) by culture-independent analysis poses specific challenges due to its highly variable bacterial density. Approach: We developed a methodology to analyze low- density BALF using a serially diluted mock community and BALF from uninfected LTR. Methods/Results: A mock microbial community was used to establish the properties of true- positive taxa and contaminants in BALF. Contaminants had an inverse relationship with input bacterial density. Concentrating samples increased the bacterial density and the ratio of community taxa (signal) to contaminants (noise), whereas DNase treatment decreased density and signal:noise. Systematic removal of contaminants had an important impact on microbiota-inflammation correlations in BALF. Conclusions: There is an inverse relationship between microbial density and the proportion of contaminants within microbial communities across the density range of BALF. This study has implications for the analysis and interpretation of BALF microbiota. ii Acknowledgments To my supervisor, Dr. Bryan Coburn, I am eternally grateful for the opportunity you have given me by accepting me as your graduate student. Over the past two years working with you has been both rewarding and at times very challenging. Thank you for guiding me in the right direction, being as honest as possible, pushing me to do my best and most of all being a supportive mentor throughout my master’s degree. The lessons you have taught me not only has made me a better scientist but a better person and I will continue to use them throughout my future career. To my lab mates, especially Saumya Bansal who has endured this whole process with me from start to finish and Ashley Rooney for being my coffee mate/great listener, thank you for being a great support system. I really do value you all of your help and have grown to appreciate you all as my science family! To my advisory committee, Dr. Tereza Martinu, Dr. Stephen Juvet and Dr. David Hwang, thank you all for continued guidance throughout these two years. Your expertise in the lung transplantation and pathology has allowed me to gain a greater understanding of this field. To our collaborators at the Toronto Lung Transplant Program and within the Martinu lab, I would like to thank you for your work collecting samples, diagnosing patients and for the cytokine analysis presented in this thesis. A special thank you to Dr. Liran Levey, for collecting all the clinical data and the cytokine analysis and Dr. Pierre Schneeberger and Dr. Youngho Lee for collaborating on the analysis of 16S data and qPCR analysis of lung transplant samples To my family, I want to thank you for being the amazing supportive people who have relentlessly encouraged me to pursue my academic career. Words cannot express the appreciation I have for your unconditional support and the sacrifices you have made to help me achieve my goals. iii Table of Contents Acknowledgments .......................................................................................................................... iii Table of Contents ........................................................................................................................... iv List of Tables .................................................................................................................................. v List of Figures ................................................................................................................................ vi List of Abbreviations and Definitions .......................................................................................... viii Chapter 1 Introduction .................................................................................................................... 1 1.1 Human microbiome ........................................................................................................ 1 1.2 Airway microbiome ........................................................................................................ 3 1.3 Allograft microbiota in lung transplant recipients .......................................................... 8 1.4 Chronic lung allograft dysfunction(CLAD) versus allograft microbiota ..................... 13 1.5 Limitations with allograft microbiota research to date ................................................. 17 1.6 Rationale ....................................................................................................................... 22 Chapter 2 Materials and Methods ................................................................................................. 23 2.1 Optimization of low density samples with serially mock community .......................... 23 2.2 Optimization of low density BALF samples from lung transplant recipients with CLAD 30 Chapter 3 Results .......................................................................................................................... 33 3.1 Mock community serial dilution ................................................................................... 33 3.2 Pre-sequencing treatments ............................................................................................ 48 3.3 Post sequencing removal of contaminants .................................................................... 56 3.4 Removal of contaminants from lung transplant cohort ................................................. 63 3.5 Threshold of 16S rRNA sequencing detection ............................................................. 75 Chapter 4 Discussion .................................................................................................................... 77 4.1 Study limitations ........................................................................................................... 78 4.2 Comparison to published reports .................................................................................. 80 4.3 Future Directions .......................................................................................................... 83 References ..................................................................................................................................... 87 iv List of Tables Table 1. List of contaminating taxa from mock community serial dilutions ................................ 37 Table 2.Contaminants that were reproducible within more than one density of mock community. ....................................................................................................................................................... 47 Table 3. Contaminants that were retained post-filtration in mock communities .......................... 61 Table 4. Patient characteristics ..................................................................................................... 68 v List of Figures Figure 1. A representation of the relationship between contaminants, microbial community complexity and density during 16S rRNA sequencing. ................................................................ 20 Figure 2. Overview of pre-sequencing optimization of low density samples ............................... 29 Figure 3.Range of absolute microbial densities within allograft microbiota of LTR. .................. 34 Figure 4. Overview of negative control collection during the processing of mock community samples. ......................................................................................................................................... 35 Figure 5. Sequence profile of serially diluted mock community samples .................................... 36 Figure 6. Overlapping taxa from mock community contaminants and reagents .......................... 39 Figure 7. Heat map of the relative abundance of taxa within each sample clustered by Bray- Curtis dissimilarity. ....................................................................................................................... 40 Figure 8. Source tracker 2 proportions for mock community samples ......................................... 41 Figure 9. Bray-Curtis Dissimilarity between mock community samples to the highest density mock community ( 107CFU/mL). ................................................................................................. 44 Figure 10. Reproducibility of technical replicates of the serially diluted mock community samples .......................................................................................................................................... 45 Figure 11. Relative abundance of all contaminants in mock community samples ....................... 46 Figure 12. Quantitative results of pre-sequencing treatment on mock community samples using 16S rRNA qPCR. .......................................................................................................................... 52 Figure 13. Change in relative abundance of mock taxa and contaminants after
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