Development of an integrated omics in silico workflow and its application for studying bacteria-phage interactions in a model microbial community

Microbial communities are ubiquitous and dynamic systems that inhabit a multitude of environments. They underpin natural as well as biotechnological processes, and are also implicated in human health. The elucidation and understanding of these structurally and functionally complex microbial systems using a broad spectrum of toolkits ranging from in situ sampling, high-throughput data generation ("omics"), bioinformatic analyses, computational modelling and laboratory experiments is the aim of the emerging discipline of Eco-Systems Biology. Integrated workflows which allow the systematic investigation of microbial consortia are being developed. However, in silico methods for analysing multi-omic data sets are so far typically lab-specific, applied ad hoc, limited in terms of their reproducibility by different research groups and suboptimal in the amount of data actually being exploited. To address these limitations, the present work initially focused on the development of the Integrated Meta-omic Pipeline (IMP), a large-scale reference-independent bioinformatic analyses pipeline for the integrated analysis of coupled metagenomic and metatranscriptomic data. IMP is an elaborate pipeline that incorporates robust read preprocessing, iterative co-assembly, analyses of microbial community structure and function, automated binning as well as genomic signature-based visualizations. The IMP-based data integration strategy greatly enhances overall data usage, output volume and quality as demonstrated using relevant use-cases. Finally, IMP is encapsulated within a user-friendly implementation using Python while relying on Docker for reproducibility. The IMP pipeline was then applied to a longitudinal multi-omic dataset derived from a model microbial community from an activated sludge biological wastewater treatment plant with the explicit aim of following bacteria-phage interaction dynamics using information from the CRISPR-Cas system. This work provides a multi-omic perspective of community-level CRISPR dynamics, namely changes in CRISPR repeat and spacer complements over time, demonstrating that these are heterogeneous, dynamic and transcribed genomic regions. Population-level analysis of two lipid accumulating bacterial species associated with 158 putative bacteriophage sequences enabled the observation of phage-host population dynamics. Several putatively identified bacteriophages were found to occur at much higher abundances compared to other phages and these specific peaks usually do not overlap with other putative phages. In addition, there were several RNA-based CRISPR targets that were found to occur in high abundances. In summary, the present work describes the development of a new bioinformatic pipeline for the analysis of coupled metagenomic and metatranscriptomic datasets derived from microbial communities and its application to a study focused on the dynamics of bacteria-virus interactions. Finally, this work demonstrates the power of integrated multi-omic investigation of microbial consortia towards the conversion of high-throughput next-generation sequencing data into new insights. iii SCIENTIFIC OUTPUT Major parts of this thesis are based upon work that has either been published, is currently under peer-review and/or ready for submission with the candidate as the first author. In addition, the candidate has also coauthored several publications of which minor parts are incorporated in the thesis. The full list of scientific outputs is listed in sections below and the original manuscripts are provided in the Appendix A. Publications in peer-review journals • Shaman Narayanasamy, Emilie E.L. Muller, Abdul R. Sheik, Paul Wilmes (2015). Integrated omics for the identification of key functionalities in biological wastewater treatment microbial communities. Microbial Biotechnology 8: 363-368. [Appendix A.1] • Shaman Narayanasamy†, Yohan Jarosz†, Emilie E.L. Muller, Anna Heintz-Buschart, Malte Herold, Anne Kaysen, Cédric C. Laczny, Nicolàs Pinel, Patrick May, Paul Wilmes (2016) IMP: a pipeline for reproducible reference-independent integrated metagenomic and metatranscriptomic analyses. Genome Biology 17: 260. [Appendix A.2] • Emilie E.L. Muller, Nicolás Pinel, Cédric C. Laczny, Michael R. Hoopmann, Shaman Narayanasamy, Laura A. Lebrun, Hugo Roume, Jake Lin, Patrick May, Nathan D. Hicks, Anna Heintz-Buschart, Linda Wampach, Cindy M. Liu, Lance B. Price, John D. Gillece, Cédric Guignard, Jim M. Schupp, Nikos Vlassis, Nitin S. Baliga, Robert L. Moritz, Paul S. Keim, Paul Wilmes (2014). Community-integrated omics links dominance of a microbial generalist to fine-tuned resource usage. Nature Communications 5: 5603. [Appendix A.3] • Hugo Roume, Anna Heintz-Buschart, Emilie E.L. Muller, Patrick May, Venkata P. Satagopam, Cédric C. Laczny, Shaman Narayanasamy, Laura A. Lebrun, Michael R. Hoopmann, Jim M. Schupp, John D. Gillece, Nathan D. Hicks, David M. Engelthaler, Thomas Sauter, Paul S. Keim, Robert L. Moritz, Paul Wilmes (2015). Comparative integrated omics: identification of key functionalities in microbial community-wide metabolic networks. NPJ Biofilms and Microbiomes 1: 15007. [Appendix A.4] †Co-first author iv Submissions in peer-review journals • Linda Wampach, Anna Heintz-Buschart, Angela Hogan, Emilie E.L. Muller, Shaman Narayanasamy, Cédric C. Laczny, Luisa W. Hugerth, Lutz Bindl, Jean Bottu, Anders F. Andersson, Carine de Beaufort, Paul Wilmes (submitted). Colonization and succession within the human gut microbiome by archaea, bacteria and microeukaryotes during the first year of life. Frontiers in Microbiology [Appendix A.5] • Anne Kaysen, Anna Heintz-Buschart, Emilie E. L. Muller, Shaman Narayanasamy, Linda Wampach, Cédric C. Laczny, Katharina Franke, Jörg Bittenbring, Jochen G. Schneider, Paul Wilmes (submitted). Integrated meta-omic analyses of the gastrointestinal tract microbiome in patients undergoing allogeneic stem cell transplantation. Journal of Experimental & Clinical Cancer Research [Appendix A.6] Publication in non-peer review platform • Shaman Narayanasamy†, Yohan Jarosz†, Emilie E.L. Muller, Cédric C. Laczny, Malte Herold, Anne Kaysen, Anna Heintz-Buschart, Nicolàs Pinel, Patrick May, Paul Wilmes (2016). IMP: a pipeline for reproducible metagenomic and metatranscriptomic analyses. BioRxiv. [Appendix A.8] Manuscripts in preparation • Emilie E.L. Muller†, Shaman Narayanasamy†, Myriam Zeimes, Laura A. Lebrun, Nathan D. Hicks, John D. Gillece, James M. Schupp, Paul Keim, Paul Wilmes (in preparation). First draft genome sequence of a strain belonging to the Zoogloea genus and its gene expression in situ. [Appendix A.7] • Shaman Narayanasamy, Emilie E. L. Muller, Laura A. Lebrun, Nathan D. Hicks, John D. Gillece, James M. Schupp, Paul S. Keim, Paul Wilmes (in preparation). The dynamics of bacteriophages and bacterial host populations within oleaginous microbial consortia within wastewater treatment plants. [Chapter 3] Oral presentations in scientific conferences, symposia and workshops • Dynamic changes in the CRISPR-spacer complement and targeted bacteriophages within a natural microbial community resolved using time-resolved metagenomics and metatranscriptomics (2014). Opening the Microbial World With Metagenomics. Helsinki, Finland. • Metagenomic and metatranscriptomic analyses of CRISPR-Cas dynamics within a microbial community (2015). Life Sciences PhD days. Belval, Luxembourg. • Integrated omics provides unprecedented insights into microbial community structure and function (2016) European Space Agency workshop for Micro-Ecological Life Support Systems Alternative (MELiSSA). Lausanne, Switzerland. †Co-first author v • Metagenomic and metatranscriptomic analyses of CRISPR-Cas dynamics within a mixed microbial community (2016). 16th Conference of the International Society of Microbial Ecology. Montreal, Canada. • IMP: A reproducible pipeline for reference-independent integrated metagenomic and metatranscriptomic analyses (2016). LCSB Minisymposium on Lab Automation Belval, Luxembourg. • IMP: The Integrated Meta-omic Pipeline: a tale of analysis and automation towards reproducible research results (2016). 1st RSG Luxembourg Congress & 2nd BeNeLuxFr Symposium Belval, Luxembourg. Poster presentations in scientific conferences, symposia and workshops • A dynamic population-level model of antiviral defense mechanisms (2013). Life Science PhD Days. Luxembourg, Luxembourg. • Eco-systems biology of microbial communities: Integration of biomolecular information from unique samples (2013). EMBL Symposium: New Approaches and Concepts in Microbiology. Heidelberg, Germany. • Eco-systems biology of microbial communities: Integration of biomolecular information from unique samples (2013). 2nd Symposium of Systems Biomedicine. Belval, Luxembourg. vi CONTENTS Abstract iii Scientific output iv