AN INTEGRATED APPROACH TO ENHANCING FUNCTIONAL ANNOTATION OF SEQUENCES FOR DATA ANALYSIS OF A TRANSCRIPTOME Matthew Morritt Hindle, BSc.(Hons) MSc. Supervisors Professor Christopher J. Rawlings Biomathematics and Bioinformatics, Rothamsted Research Dr Dimah Z. Habash Plant Science, Rothamsted Research Professor Charlie Hodgman Multidisciplinary Centre for Integrative Biology, The University of Nottingham This thesis was submitted to The University of Nottingham for the degree of Doctor of Philosophy July 2012 Abstract Given the ever increasing quantity of sequence data, functional annotation of new gene sequences persists as being a significant challenge for bioinformatics. This is a particular problem for transcriptomics studies in crop plants where large genomes and evolutionarily distant model organisms, means that identi- fying the function of a given gene used on a microarray, is often a non-trivial task. Information pertinent to gene annotations is spread across technically and semantically heterogeneous biological databases. Combining and exploit- ing these data in a consistent way has the potential to improve our ability to assign functions to new or uncharacterised genes. Methods: The Ondex data integration framework was further developed to integrate databases pertinent to plant gene annotation, and provide data infer- ence tools. The CoPSA annotation pipeline was created to provide automated annotation of novel plant genes using this knowledgebase. CoPSA was used to derive annotations for Affymetrix GeneChips available for plant species. A conjoint approach was used to align GeneChip sequences to orthologous pro- teins, and identify protein domain regions. These proteins and domains were used together with multiple evidences to predict functional annotations for se- quences on the GeneChip. Quality was assessed with reference to other annota- tion pipelines. These improved gene annotations were used in the analysis of a time-series transcriptomics study of the differential responses of durum wheat varieties to water stress. Results and Conclusions: The integration of plant databases using the On- dex showed that it was possible to increase the overall quantity and quality of information available, and thereby improve the resulting annotation. Direct data aggregation benefits were observed, as well as new information derived from inference across databases. The CoPSA pipeline was shown to improve coverage of the wheat microarray compared to the NetAffx and BLAST2GO pipelines. Leverage of these annotations during the analysis of data from a transcriptomics study of the durum wheat water stress responses, yielded new biological insights into water stress and highlighted potential candidate genes that could be used by breeders to improve drought response. i Acknowledgements This thesis was only possible through of the work of many colleagues at Rothams- ted Research. My thanks to all the members of the TRITIMED and Ondex SABR projects, whose contributions I acknowledge by references within this work. "We are like dwarfs on the shoulders of giants, so that we can see more than they, and things at a greater distance, not by virtue of any sharpness of sight on our part, or any physical distinction, but because we are carried high and raised up by their giant size" Bernard of Chartres I am indebted to my supervisors over the last four years: Chris Rawlings, Dimah Habash, Jacob Köhler, and Charlie Hodgman. I particularly wish to thank Marcela Baudo, who together with Dimah Habash conceived and ex- ecuted the time-series experiment that made this work possible. Michael Defoin- Platel provided invaluable collaboration and advice in the evaluation and re- finement of CoPSA. I would also like to thank my fellow developers, who con- tributed elements of Ondex and ideas that were built upon or used during this research. I am pleased to acknowledge Andrea Splendiani, Artem Lysenko, Berend Hoekman, Catherine Canevet, Jan Taubert, Keywan Hassani-Pak, Mat- thew Pocock, and Rainer Winnenburg. I also acknowledge Mansoor Saqi for his suggestions throughout this project and Stephen Powers for his statistical advice, and contributions to the analysis of TRITIMED data. ii Definitions of terms and abbreviations AAO [Gene] Aldehyde Oxidase: a gene that encodes an enzyme that catalyzes the final step of ABA biosynthesis. ABA [Phytohormone] Abscisic acid: one of the major plant hormones that forms a hub in a signalling network which regulates many cellular and plant wide processes. ABAR/CHLH [Gene] cMagnesium-protoporphyrin IX chelatase H subunit: A protein that was put forward by Shen et al. (2006) as a candidate ABA receptor. ABI [Gene Family] A family of ABA insensitive gene loci. Accession [Bioinformatics term] A unique sequence of characters that within the scope of some definition uniquely define a database entry, independent of a database instance. If multiple accessions are merged together, then a new ac- cession is created and the previous made obsolete. Only one accession should ever identify an entry, where obsolete accessions are present in an entry, this is sometimes referred to as the primary accession. ANOVA [Statistical methodology] ANalysis Of VAriance: a statistical procedure by which observations are partitioned into groups representing sources of vari- ation. Application Programming Interface(s) (API) [[Computer Science term]A defined set of publicly exposed functions in a program, that allow another pro- gram to make use of its resources. A good API exposes required functionality, while minimising complexity. BiFC [Molecular biology methodology] Biomolecular florescence complimentation (BiFC): A methodology for confirming protein-protein interactions. Florescent protein fragments are attached to two or more proteins suspected of interact- ing. Interaction of these proteins will cause the fragments to reform and emit its florescence colour, thereby confirming the interaction. CDPK [Protein] Calcium Dependent Protein Kinase: a group of proteins that are stimulated by Ca2+ to initiate their activity. Drought Stress [Agricultural Plant Science term] The limitation on maximum po- tential crop yield imposed by a water limitation. Endoplasmic Reticulum (ER) [Cell organelle] An organelle that forms an inter- connected network of tubules, vesicles, and cisternae within a eukaryote cell. It is mainly involved in protein, lipid and steroid synthesis, and carbohydrate iii and steroid metabolism. Extensible Mark-up Language (XML) [File format] A structured text document, conforming to rules defined by W3C (2011). It allows documents to be machine- readable. FCA [Gene] A gene encoding a posttranscriptional regulator of tran- scripts involved in the flowering process (Macknight et al., 1997). G protein [Protein family] A family of proteins that bind to guanine nucleotides (GTP and GDP), and are involved in signalling. GFP-florescence [Molecular Biology methodology]: Florescence labelling using a small (238 amino acids) Green Florescence Protein (GFP), which emits green light when exposed to blue light. The GFP gene can be fused into a target gene, which then may express a protein with florescence. The target genes localisa- tion and expression in the cell can therefore be monitored (Phillips, 2001). GPA1 [Gene] The sole member of the G-protein Ga-subunit family within the Arabidopsis genome. GTG [Protein family] GPCR-type G proteins (GTG): A gene family including GTG1 and GTG2 which are candidate ABA binding proteins (Pandey et al., 2009). Homolog(y) [Genetics term] The relationship between two genes that are des- cended from a common ancestral genes (Fitch, 2000). HSF [Protein family] Heat Shock Factors (HSFs). A family of transcription factors which regulate HSPs. HSP [Protein family] Heat Shock Proteins (HSPs). A family of proteins that as- sist as chaperones in protein folding (Hu et al., 2009). NAC [Protein family] A superfamily of transcription factors, many of which are involved in hormonal regulation (Jensen et al., 2010). NCED [Gene family]A gene family encoding 9-cis-epoxycarotenoid dioxygenase, which is an enzyme that is part of the ABA biosynthesis pathway. MAPK [Protein] Mitogen-Activated Protein Kinase: a signalling molecule that forms the initial activator of the MAPK cascade. The second and third elements of this cascade are MAPK Kinase (MAPKK) and MAPKK Kinase (MAPKKK), respectively. MYB [Protein family] A superfamily of transcription factors, which has the largest number of members in Arabidopsis (Yanhui et al., 2006). They commonly in- volved in the regulation of developmental processes and defence response. Object-Oriented (OO) [Computer Science term] A programming paradigm based around data objects, which consist of a group of fields and methods. OO is associated with a number of good practices. Encapsulation: access to an ob- iv ject is restricted by public and private components. Abstraction: concepts or ideas independent of an implementation instance are separated. Modularity: where possible software is composed of separate interchangeable components. Inheritance: common properties and methods of objects are shared between implementations (e.g. Java class inheritance). Polymorphism: the ability to cre- ate variable, functions or objects that have multiple implementation forms (e.g. Java interfaces). Ortholog(y) [Genetics term] The relationship between two homologous genes, whose common ancestor is the last universal common ancestor of the taxa from which the two sequences were obtained (Fitch, 2000). OXL [File format] An