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Development of Genome-Wide Genetic Assays In DEVELOPMENT OF GENOME-WIDE GENETIC ASSAYS IN DESULFOVIBRIO VULGARIS HILDENBOROUGH A Dissertation presented to the Faculty of the Graduate School at the University of Missouri In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy By SAMUEL R. FELS Dr. Judy Wall, Dissertation Supervisor MAY 2015 © Copyright by Samuel Fels 2015 All Rights Reserved The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled Development of genome-wide genetic assays in Desulfovibrio vulgaris Hildenborough presented by Samuel Fels, a candidate for the degree of doctor of philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Professor Judy Wall Professor Mark McIntosh Professor George Stewart Assistant Professor Michael Baldwin Research Associate Professor Robert Schnabel Thanks Mom and Dad (for everything) Acknowledgements I would like to thank everyone who contributed to my success throughout graduate school. Judy Wall has been the best mentor and academic advisor anyone could ask for, and this research would not have been possible without her knowledge and foresight. All members of the Wall lab added something to this research, but very significant contributions were made by Grant Zane, Kimberly Keller, Barbara Giles, Hannah Korte, Geoff Christenson, and Kara de Leon. I would also like to thank members of the MU DNA Core including Sean Blake, Nathan Bivens, and Karen Bromert for their help in analyzing and troubleshooting many aspects of DNA sequencing. Minyong Chung of the Vincent J. Coates Genomic Sequencing Laboratory in Berkeley, California also provided valuable insight and sequencing capacity to this thesis. Many collaborators through the Department of Energy’s ENIGMA group also provided key insights, including Morgan Price, Adam Arkin, Adam Deutschbauer, and Steve Brown of Lawrence Berkeley National Laboratory. My graduate research would not have been possible without support from my family and friends, most notably from my parents and my sister/roommate Becca Fels who agreed to put up with me over the last eight months. Constant mental support and wonderful rainbow sparkles were provided by the amazing Rashaun Wilson. This research was funded by Ecosystems and Networks Integrated with Genomes and Molecular Assemblies (ENIGMA), Office of Science, OBER, U.S. Department of Energy, Contract No. DE-AC02-05CH11231 and a supplemental ENIGMA Discovery Grant for 3’ RNA-seq, which I wrote and received funding for. ii Table of Contents Acknowledgements……………………………………………..….…………….………………………………………………ii List of Tables…………………………………….……………………………….…………………………………….…………….v List of Figures………………………………………………………………….…………………………………………………….vi List of Appendices………………………………………………………….………………………………….…………………viii Abstract……………………………………………………………………….………………………………………..……………..ix Chapter 1 – Introduction……………………………………………………………………………………………..….…….1 1.1 History of Desulfovibrio vulgaris Hildenborough………………………………………....…..…….1 1.2 Motivation for this work……………………………………………………………………………….…..……4 Chapter 2 – Optimizing transposon insertion in DvH…………………………………………….………….…….7 2.1 Introduction…………………………………………………………………………..………………….…..……….7 2.2 Mutagenesis via electroporation……………………………………………………………………..……..9 2.3 Mutagenesis via conjugation………………………………………………………………….………..…….11 2.4 Materials and Methods………………………………………………………………………….….…………..12 2.5 Results…………………………………………………………………………………………..…………………..…..15 Chapter 3 – Development of TnLE-seq library preparation techniques.………………………..……….17 3.1 Introduction……………………………………………………………………………………………….…………..17 3.2 Development of a liquid enrichment protocol………………………………………………………..21 3.3 Changes to an existing Illumina™ library preparation technique…………………..………..24 3.4 Materials and Methods…………………………………………………………………………………..……..29 3.5 Results……………………………………………………………………………………………………………………33 Chapter 4 – Creation of an informatics pipeline and statistical analysis of TnLE-seq data..…….37 4.1 Introduction………………………………………………………………………………………………..…….……37 4.2 Development of the TnLE-seq computational pipeline…………………………………...……..39 4.3 Visualization of insertion points and read counts across a bacterial genome….……...43 4.4 Statistical analysis to determine essential genes……………………….……….…………..………44 4.5 Statistical analyisis of changes in gene fitness………………………………………………………..46 4.6 Materials and Methods………………………………………………………………..………..………………47 4.7 Results……………………………………………………………………………………………………..…………….52 iii Chapter 5 – Isolation of high-quality RNA from bacterial cultures……………………………….………..66 5.1 Introduction………………………………………………………………………………………………..………….66 5.2 Use of a standard RNA quality metric……………………………………………………………………..67 5.3 RNA isolation options……………………………………………………………………………………………..68 5.4 Rho and PNPase deletions and potential RNA quality impact……………..………………….70 5.5 Materials and Methods……………………………………..………………………………………….……….74 5.6 Results……………………………………………………………………………………………………….….……….76 Chapter 6 – Development of the 3’ RNA-seq method to determine transcript end sites….…….84 6.1 Introduction…………………………………………………………………………………………………...……..84 6.2 Ribosomal RNA depletion…………………………………………………………………………..…….……88 6.3 Selective ligation of a known DNA aptamer to 3’RNA ends…………….…………….….…….90 6.4 First and second strand cDNA synthesis……………………………………………………….………..92 6.5 Exonuclease I treatment and PCR amplification of the final library…………..……………94 6.6 Methods………………………………………………………………………………………………………………..97 6.7 Results………………………………………………………………………………………………………………….102 Chapter 7 – Analysis of differential expression by total RNA-seq…………..…………………………...108 7.1 Introduction…………………………………………………………………………………….…………………..108 7.2 Potential impact of pnp deletion on transcript expression…………………….……………..110 7.3 Materials and Methods……………………………………………………………..…………………………112 7.4 Results……………………………………………………………………………………………….…………………114 Chapter 8 – Contributions to the field and future directions…………………………..…………………..120 8.1 Contributions to the field………………………………………………………………………….………….120 8.2 Future Directions…………………………………………………………………………………..……………..121 Bibliography…………………………………………………………………………………………………….………………...123 Vita………………………………………………………………………………………………………………………………….…263 iv List of Tables Table 1 – Population tracking by colony counts, performed before and after mutant generation………………………………………………………………………………………………………………………………..15 Table 2 – Impact of oxidation stress on DvH gene products……………………………………………………..18 Table 3 – CFU tracking, 1:10 vs 1:33 dilution…………………………………………………………………………….33 Table 4 – Read quality summary……………………………………………………………………………………………….48 Table 5 – Alignment/coverage summary…………………………………………………………………………………..48 Table 6 – Essential genes…………………………………………………………………………………………………….…….54 Table 7 – Gene fitness changes between experiments………………………………………………………………59 Table 8 – Genes with highest fitness in the presence of 100 mM nitrate……………………..…………..62 Table 9 – Total RNA-seq library information……………………………………………………………..…………….114 Table 10 – Control and experimental comparisons………………………………………………….……………..115 v List of Figures Figure 1 – Transmission electron micrograph of a single DvH bacterium…………………………………….1 Figure 2 – Anaerobic glove bag used for growth and manipulation of DvH………………………………...2 Figure 3 – Chromosome-wide map of cataloged transposon mutant library constructed in DvH..3 Figure 4 – Structure of pRL27 and the final transposon in the DvH genome……………..………………..8 Figure 5 – Comparison of mutagenesis and enrichment in two different Tn-seq experimental approaches…………………………………………………………………………………………………………………………….…24 Figure 6 – Illumina® Library Preparation (part 1)…………………………………………….………………………..25 Figure 7 – Illumina® Library Preparation (part 2).………………………………………………………….……….…27 Figure 8 – Formula to obtain the gene fitness value for any gene……………………..………..……………37 Figure 9 – Gamma distributions to model essential gene probability……………………………………….44 Figure 10 – Cumulative probability to call essential vs non-essential………………………………..………45 Figure 11 – Two-condition comparison of all gene fitness values……………………………………………..46 Figure 12 – Generation of gene fitness values from aligned reads………………………………….………..49 Figure 13 – Sample histogram for fitting gamma distributions……………………………………..…………..50 Figure 14 – Example of read abundance mapping across the DvH genome…………………….………..53 Figure 15 – Gene fitness comparison of rich media biological replicates…..………………………………56 Figure 16 – Gene fitness comparison of rich and minimal media……………………….……………………..58 Figure 17 – Gene fitness comparison of WT DvH and JW710………………………………………..………….60 Figure 18 – Gene fitness comparison of JW710 with and without inhibitory nitrate…………………61 Figure 19 – Gene fitness comparison of JW710 and JW3319………………………………………………..…..64 Figure 20 – Representative Fragment Analyzer™ electrophoretograms used to calculate RQN…70 Figure 21 – Growth comparison of deletion mutant strains……………………………………………..……….78 Figure 22 – Average RQN scores by DvH strain………………………………………………………….………………79 Figure 23 – Average RNA Yield by DvH Strain…………………………………………………………………..……….80 Figure 24 – Comparison of total RNA-seq with simultaneous 5’ and 3’ RNA-seq……………..………..82 Figure 25 – Summary of the 5’ RNA-seq library preparation protocol………………………………..……..86 Figure 26 – Nucleic acid fragments before and after 3’ RNA-seq library preparation………….….…87 Figure 27 – AIR™ Ligase mediated attachment of adenylated DNA fragment to 3’ mRNA ends..91 Figure 28 – First- and second-strand cDNA synthesis……………………………………………………………….93
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