UCC Library and UCC researchers have made this item openly available. Please let us know how this has helped you. Thanks! Title Genetic analysis of bacteriophages from clinical and environmental samples Author(s) Knapik, Kamila Z. Publication date 2013 Original citation Knapik, K. Z. 2013. Genetic analysis of bacteriophages from clinical and environmental samples. PhD Thesis, University College Cork. Type of publication Doctoral thesis Rights © 2013, Kamila Z. Knapik http://creativecommons.org/licenses/by-nc-nd/3.0/ Item downloaded http://hdl.handle.net/10468/1380 from Downloaded on 2021-10-04T07:46:11Z Genetic analysis of bacteriophages from clinical and environmental samples University College Cork Coláiste na hOllscoile Corcaigh A Thesis presented to the National University of Ireland for the Degree of Doctor of Philosophy by Kamila Knapik Department of Microbiology National University of Ireland Cork Head of Department: Prof. Gerald F. Fitzgerald Supervisor: Prof. Michael Prentice July 2013 Table of Contents Declaration ................................................................................................................. vi Acknowledgements ................................................................................................... vii List of Tables ........................................................................................................... viii List of Figures ............................................................................................................. x List of Abbreviations .............................................................................................. xiii Abstract ..................................................................................................................... xv Chapter 1: ................................................................................................................... 1 Literature review ........................................................................................................ 1 1.1. Introduction to bacteriophages ...................................................................... 2 1.1.1. Phage discovery and research ................................................................ 2 1.1.2. Phage structure and classification .......................................................... 3 1.1.3. Phage life cycles ..................................................................................... 7 1.1.4. Phage applications and significance....................................................... 8 1.1.5. Phage abundance and diversity ............................................................ 10 1.2. Introduction to metagenomics ..................................................................... 15 1.2.1. Viral metagenomics ............................................................................. 15 1.2.2. Specific applications of viral metagenomics........................................ 20 1.2.3. Methods for generating viral metagenomes ......................................... 23 1.3. Introduction to next-generation sequencing and bioinformatics .................. 29 1.3.1. Sequencing strategies ........................................................................... 29 1.3.2. Computation approaches for characterizing sequenced viral metagenomes ...................................................................................................... 32 1.4. Objectives of this study ............................................................................... 38 Chapter 2: ................................................................................................................. 39 Metagenomic sequencing of DNA viruses in cystic fibrosis sputum ................... 39 Abstract ..................................................................................................................... 40 2.1. Introduction ................................................................................................. 41 2.2. Materials and Methods ................................................................................ 43 2.2.1. Sample collection ................................................................................. 43 2.2.2. Viral particle purification ..................................................................... 43 2.2.3. DNA isolation and sequencing............................................................. 44 2.2.4. Sequence processing ............................................................................ 44 ii 2.2.5. Assembly of sequence reads ................................................................ 45 2.2.6. Sequence annotation............................................................................. 45 2.2.7. Metagenome diversity .......................................................................... 46 2.2.8. Metagenome comparison ..................................................................... 46 2.2.9. Antibiotic resistance genes ................................................................... 47 2.2.10. Virulence genes .................................................................................. 47 2.2.11. CRISPR spacer analyses .................................................................... 47 2.2.12. PCR validation of contigs .................................................................. 48 2.2.13. TTV phylogenetic analysis ................................................................ 49 2.3. Results ......................................................................................................... 50 2.3.1. Taxonomic classification of reads ........................................................ 50 2.3.2. Assembly characteristics ...................................................................... 50 2.3.3. Diversity of viruses in CF sputum ....................................................... 57 2.3.4. Functional classification....................................................................... 57 2.3.5. Resistance to antibiotics ....................................................................... 64 2.3.6. Toxin genes .......................................................................................... 64 2.3.7. Comparison with other viral metagenomes.......................................... 68 2.3.8. CRISPR sequences in CF viral metagenome ....................................... 69 2.3.9. Identification of novel Torque Teno viruses in CF sputum ................. 69 2.4. Discussion ................................................................................................... 74 Chapter 3: ................................................................................................................. 79 Metagenomic sequencing of DNA viruses in dairy wastewater ........................... 79 Abstract ..................................................................................................................... 80 3.1. Introduction ................................................................................................. 81 3.2. Material and Methods .................................................................................. 83 3.2.1. Sample collection ................................................................................. 83 3.2.2. Viral particles purification ................................................................... 83 3.2.3. DNA isolation and sequencing............................................................. 83 3.2.4. Confocal microscopy ........................................................................... 84 3.2.5. Electron microscopy............................................................................. 85 3.2.6. Sequence processing ............................................................................ 85 3.2.7. Assembly of sequence reads ................................................................ 85 3.2.8. Sequence annotation............................................................................. 86 iii 3.2.9. Analysis of complete genomes............................................................. 86 3.2.10. Phylogenetic analyses ........................................................................ 87 3.2.11. Metagenome diversity ........................................................................ 87 3.2.12. Metagenome comparison ................................................................... 87 3.2.13. Antibiotic resistance genes ................................................................. 87 3.2.14. CRISPR spacer analyses .................................................................... 88 3.2.15. Microbial diversity in activated sludge ................................................. 88 3.3. Results ......................................................................................................... 90 3.3.1. Visualization of viruses in activated sludge ......................................... 90 3.3.2. Sequence reads taxonomic classification ............................................. 92 3.3.3. Contig assembly and taxonomic classification .................................... 97 3.3.4. Identification of novel ssDNA circular viruses .................................... 99 3.3.5. Sequence analysis of the largest assembled contig resembling Myoviridae ........................................................................................................ 106 3.3.6. Diversity of viruses in activated sludge ............................................. 108 3.3.7. Functional