VIRUS DETECTION IN A METAGENOMIC SEQUENCE DATASET: METHODS AND APPLICATIONS By ANTHONY STOBBE Bachelor of Science in Health Science Southwestern Oklahoma State University Weatherford, Oklahoma 2006 Master of Science in Biochemistry Oklahoma State University Stillwater, Oklahoma 2012 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY July, 2013 VIRUS DETECTION IN A METAGENOMIC SEQUENCE DATASET: METHODS AND APPLICATIONS Dissertation Approved: Ulrich Melcher Dissertation Adviser Patricia Canaan Peter Hoyt Jacqueline Fletcher William Schneider ii ACKNOWLEDGEMENTS I would like to extend my deepest thanks to my major professor and mentor, Ulrich Melcher. His passion for science and discovery was one reason I joined his laboratory, and remained a source of motivation for me over these past four years. I would also like to thank each of my committee members. Bill Schneider taught me the ins and outs of the scientific community. Thank you for support and confidence boosts when they were needed. Jacqueline Fletcher showed me the importance of professionalism and networking. It is obvious that she cares for the future of all of the students that she interacts with. I would like to thank Patricia Canaan, who lent me my first book on writing scripts, setting me down the path of bioinformatics, as well as Peter Hoyt whose continued efforts to make Oklahoma State a center for bioinformatics in Oklahoma has led to great experiences for the future bioinformaticians. While my committee made sure I stayed on course academically, my friends and family provided great support when times were tough. They are always ready to celebrate successes, commiserate failures, and provide much needed relaxation. I cannot imagine a better group of colleagues and friends than the ones that I have made during my time at Oklahoma State University. I must give a special thank you to my long suffering wife, who has listened to me when I needed an ear, talked to me when I needed to listen, and has loved me when it was difficult to do so. I love you dearly. iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: ANTHONY STOBBE Date of Degree: JULY, 2013 Title of Study: VIRUS DETECTION IN A METAGENOMIC SEQUENCE DATASET: METHODS AND APPLICATIONS Major Field: BIOCHEMISTRY AND MOLECULAR BIOLOGY Abstract: Global trade of plant material has increased the introduction of foreign plant viruses in nations across the world. This has led to the need for increased abilities to detect plant viruses. Surveys assessing the viral biodiversity of environments often use Next Generation Sequencing (NGS) as a tool for virus discovery. NGS has yet to be used as a diagnostic tool due to the computational and time requirements of analyzing NGS data. The purpose of this work is first to show the importance of virus discovery, and second to describe the development and validation of a bioinformatic pipeline designed to detect and identify both DNA and RNA viruses by using pathogen-specific probes to detect virus sequence signals in a metagenomic sample dataset. Finally, Chapter VI shows how the bioinformatic pipeline can be used for the detection of unknown viruses by using general probes. iv TABLE OF CONTENTS Chapter Page I. INTRODUCTION ......................................................................................................1 II. REVIEW OF LITERATURE..................................................................................11 Sanger sequencing .................................................................................................11 Roche 454 pyrosequencing ....................................................................................12 Ion Torrent .............................................................................................................13 Illumina Solexa ......................................................................................................13 Pacific Biosciences ................................................................................................14 Sequencing Errors ..................................................................................................14 Homo-oligomers ....................................................................................................14 Base calling ............................................................................................................15 Sequencing Bias .....................................................................................................15 Read Length ...........................................................................................................15 Assembly................................................................................................................16 Newbler Assembler (de novo) ...............................................................................16 Mapping Assembly ................................................................................................17 Metagenomics ........................................................................................................17 Virus Discovery .....................................................................................................18 Nucleic Acid Based Assays ...................................................................................19 Differential Centrifugation...............................................................................19 Polymerase Chain Reactions............................................................................19 Whole Genome/Transcriptome Amplification (WGA/WTA) .........................20 Double Stranded RNA Preparation ..................................................................21 Rolling Circle Amplification ...........................................................................21 Suppressive Subtractive Hybridization ............................................................21 Microarrays ......................................................................................................22 Food Safety and Biosecurity ..................................................................................23 Pathogens of Interest ..............................................................................................24 Tobamoviruses .......................................................................................................24 Begomoviruses .......................................................................................................25 Potyviruses .............................................................................................................26 v Chapter Page III. CO-DIVERGENCE AND HOST-SWITCHING IN THE EVOLUTION OF TOBAMOVIRUSES..............................................................................................40 Summary ................................................................................................................40 Introduction ............................................................................................................41 Results ....................................................................................................................44 PafMV-TGP .....................................................................................................44 Phylogenetics ...................................................................................................47 Dating ...............................................................................................................48 BaTS Analysis of Association .........................................................................49 Disscussion ............................................................................................................50 Codivergence ...................................................................................................50 Alternatives to Codivergence ...........................................................................51 Generation of Codivergence ............................................................................55 Methods..................................................................................................................56 PVBE Methods ................................................................................................56 Phylogenetic Methods ......................................................................................57 Acknowledgements ................................................................................................58 IV. E-PROBE DIAGNOSTIC NUCLEIC ACID ANALYSIS (EDNA): A THEORETICAL APPROACH FOR HANDLING OF NEXT GENERATION SEQUENCING DATA FOR DIAGNOSTICS .....................................................75 Abstract ..................................................................................................................75 Author Summary ....................................................................................................76 Introduction ............................................................................................................76 Materials and Methods ...........................................................................................79 Pathogens and Their Sequences .......................................................................79 Experimental Flow ...........................................................................................80