Exploring the microbiome of the Mediterranean sponge Aplysina aerophoba by single-cell and metagenomics Untersuchungen am Mikrobiom des Mittelmeerschwamms Aplysina aerophoba mittels Einzelzell- und Metagenomik Doctoral thesis for a doctoral degree at the Graduate School of Life Sciences Julius-Maximilians-Universität Würzburg Section: Integrative Biology Submitted by Beate Magdalena Slaby from München Würzburg, March 2017 Submitted on: ……………………………………………………… Members of the Promotionskomitee Chairperson: Prof. Dr. Thomas Müller Primary Supervisor: Prof. Dr. Ute Hentschel Humeida Supervisor (Second): Prof. Dr. Thomas Dandekar Supervisor (Third): Prof. Dr. Frédéric Partensky Date of public defense: ……………………………………………………… Date of receipt of certificates: ……………………………………………………… ii Affidavit I hereby confirm that my thesis entitled ‘Exploring the microbiome of the Mediterranean sponge Aplysina aerophoba by single-cell and metagenomics’ is the result of my own work. I did not receive any help or support from commercial consultants. All sources and / or materials applied are listed and specified in the thesis. Furthermore, I confirm that this thesis has not yet been submitted as part of another examination process neither in identical nor in similar form. Place, Date Signature iii Acknowledgements I received financial support for this thesis project by a grant of the German Excellence Initiative to the Graduate School of Life Sciences of the University of Würzburg through a PhD fellowship, and from the SponGES project that has received funding from the European Union’s Horizon 2020 research and innovation program. I would like to thank: Dr. Ute Hentschel Humeida for her support and encouragement, and for providing so many extraordinary opportunities. Dr. Thomas Dandekar and Dr. Frédéric Partensky for the supervision and a number of very helpful discussions. my current and former colleagues in the Research Unit Marine Microbiology at the Division of Marine Ecology, GEOMAR Kiel, particularly Kristina, Hannes, Martin, Bettina, Tanja, Jutta, Regine, Sabrina, Giampiero, Laura, Lucía, Kathrin, Jule, Yu-Chen, Dr. Johannes Imhoff, Ignacio, and Alvaro for their support, their friendship, and making the relocation in the middle of the PhD project not only possible, but enjoyable. all my former colleagues at the Department of Botany II in Würzburg, especially Janine, Lucas, Tine, Anni, Elli, Wilma, Moni, Andrea, Natascha, Usama, Cheng, and Ann-Janine for their friendship and support. Dr. Gabriele Blum-Oehler, Jenny Braysher, Bianca Putz, and Franz-Xaver Kober of the Graduate School of Life Sciences for answering all my questions. The US Department of Energy Joint Genome Institute, and especially Dr. Tanja Woyke, Dr. Susannah Tringe, Dr. Scott Clingenpeel and Dr. Alex Copeland for their support and sharing their expertise. The Marine Biology Station Portorož and Piran, Slovenia, for the sampling possibility. Dr. Monika Bright for her support with sampling. Dr. Laura Steindler, Ilia Burgsdorf, and Dr. Thomas Hackl for great collaborations. Dr. Frank Förster for access to the server and support. The FACS and Cell Sorting Unit Würzburg for support with FACS sorting. Dr. Autun Purser for proof-reading. My family and friends for their unconditional support and encouragement, especially my parents, my brother, my ‘moving crew’ Markus and Willi, and Dom. iv Table of contents AFFIDAVIT ......................................................................................................................... III ACKNOWLEDGEMENTS ................................................................................................ IV TABLE OF CONTENTS ..................................................................................................... V LIST OF FIGURES ........................................................................................................... VII LIST OF TABLES ............................................................................................................... IX SUMMARY ........................................................................................................................... X ZUSAMMENFASSUNG ................................................................................................... XII 1 INTRODUCTION ........................................................................................................ 14 1.1 Sponges (phylum Porifera) .................................................................................. 14 1.2 Sponge-microbe symbiosis .................................................................................. 17 1.2.1 Microbial diversity ......................................................................................... 17 1.2.2 Microbial function.......................................................................................... 18 1.3 Aplysina aerophoba ............................................................................................... 19 1.3.1 Geographic distribution and physical properties ............................................ 19 1.3.2 The A. aerophoba microbiome ...................................................................... 20 1.4 Sequence-based analyses of microbiomes .......................................................... 21 1.4.1 Recent developments in sequencing technologies ......................................... 22 1.4.2 Why short-reads fall short .............................................................................. 24 1.4.3 Long-read sequencing in metagenomics ........................................................ 24 2 MATERIAL AND METHODS................................................................................... 26 2.1 Research questions and aims .............................................................................. 26 2.2 Sponge collection and enrichment of prokaryotic cells .................................... 27 2.3 Sequencing and analysis of cyanobacterial sponge symbionts ......................... 28 2.3.1 Laboratory methods: From sample to sequence ............................................. 28 2.3.2 Bioinformatic methods: From sequencing reads to genome comparison ...... 30 2.4 Development of a hybrid assembly pipeline for PacBio long-reads and Illumina short-reads ........................................................................................................ 32 2.4.1 Simulating sequencing reads for a test dataset ............................................... 32 2.4.2 Testing assemblers and settings ..................................................................... 33 2.4.3 Comparing and evaluating of assemblies and bins ........................................ 34 2.5 Apylsina aerophoba metagenomics ..................................................................... 36 2.5.1 Laboratory methods: DNA extraction and sequencing .................................. 36 2.5.2 Bioinformatic methods: From assembly to annotation .................................. 36 2.5.3 Statistical analysis: Comparison to references and within symbionts ............ 37 v 3 RESULTS ..................................................................................................................... 40 3.1 Assessing the genome of the “Ca. Synechococcus spongiarum” group ........... 40 3.1.1 Assessment of clade F genomes from A. aerophoba ..................................... 40 3.1.2 Comparison within the “Ca. Synechococcus spongiarum” group and to free- living references ............................................................................................................. 44 3.2 PacBio-Illumina hybrid assembly pipeline development ................................. 63 3.2.1 Statistics of the tested assembly strategies ..................................................... 63 3.2.2 Comparison back to original reference genomes ........................................... 64 3.2.3 Reference-independent binning ..................................................................... 65 3.3 Binning 37 symbiont genomes from the metagenome of A. aerophoba ........... 68 3.3.1 Assessment of metagenomic DNA extraction and sequencing ...................... 68 3.3.2 Comparison of Illumina-only and PacBio-Illumina hybrid assemblies ......... 69 3.3.3 Bacterial genomes binned from hybrid assembly .......................................... 72 3.3.4 Symbiont-reference comparison .................................................................... 78 3.3.5 Within-symbiont comparison ......................................................................... 84 4 DISCUSSION ............................................................................................................... 88 4.1 “Ca. Synechococcus spongiarum” group – closely related but different in gene content ............................................................................................................................... 88 4.1.1 An optimal candidate for ‘mini-metagenomics’ ............................................ 88 4.1.2 Lifestyle evolution in cyanobacterial symbionts of sponges ......................... 89 4.2 PacBio-Illumina hybrid assembly pipeline development ................................. 97 4.3 Metagenomic bins from the microbiome of A. aerophoba reveal unity in defense but metabolic specialization .............................................................................. 98 4.3.1 Breaking new ground in assembly strategy and choice of references ............ 98 4.3.2 Unity in defense ............................................................................................