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Culture-Independent Methods and High-Throughput Sequencing Applied to Evolutionary Microbial Genomics Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1615 Culture-independent methods and high-throughput sequencing applied to evolutionary microbial genomics ANDERS E. LIND ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6214 ISBN 978-91-513-0196-9 UPPSALA urn:nbn:se:uu:diva-336794 2018 Dissertation presented at Uppsala University to be publicly examined in B22, BMC, Husargatan 3, Uppsala, Friday, 16 February 2018 at 13:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Professor Purificación López-García (Paris-Sud University, Paris, France). Abstract Lind, A. E. 2018. Culture-independent methods and high-throughput sequencing applied to evolutionary microbial genomics. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1615. 68 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-0196-9. Culture-independent methods allow us to better understand the diversity of microorganisms on earth. By omitting the culturing step, we can gain access to species previously out of our reach. To better capture and understand the diversity of microorganisms, we have developed an alternative to 16S amplicon sequencing. This method exploits the fact that the rRNA operon in most prokaryotes contains both the 16S and the 23S genes in a determined order, close to each other. Combining this with PacBio long molecule sequencing, we can now generate amplicon containing the 16S-ITS-23S operon, allowing for a stronger phylogenetic signal. Using two culture-independent methods, single-cell genomics and metagenomics, we have been able to fully sequence the genome of two archaeal endosymbionts from the genera Methanobrevibacter and Methanocorpusculum. These methanogenic archaea were isolated from the ciliates Nyctotherus ovalis and Metopus contortus. The genomic data show evidence of genome degradation, mainly through pseudogenization of genes. These genomes represent the first genomic data from archaeal endosymbionts. The endosymbiotic archaea we have sequenced live in close proximity to hydrogen producing mitochondria, or hydrogenosomes. In ciliates it has previously been shown that the hydrogenosome of N. ovalis has retained a genome, something many other ciliate hydrogenosome have not. Using genomic and transcriptomic data from seven anaerobic ciliates, we have been able to show that these ciliate have independently evolved from ancestral mitochondria, and show various degrees of genome reduction. Furthermore we have been able to shed some new light on haloarchaeal evolution by reconstructing the genomes of five species belonging to the Marine Group IV archaea. These archaea have previously been found to be closely related to the haloarchaea, however they are not halophiles. All five genomes were obtained using metagenomic binning of the publicly available TARA Oceans dataset. The genomes are all of high quality and completeness, and are used in tree-aware ancestral reconstruction, in order to try to better understand the evolutionary transition from an anaerobic methanogen to a halophile. These studies all show how culture-independent method are powerful tools in gathering genomic information about microbes we are currently unable to culture in the lab. Keywords: Single cell genomics, metagenomics, amplicon, archaea, haloarchaea, endosymbiosis, ciliate, hydrogenosome, tree reconciliation Anders E. Lind, Department of Cell and Molecular Biology, Box 596, Uppsala University, SE-75124 Uppsala, Sweden. © Anders E. Lind 2018 ISSN 1651-6214 ISBN 978-91-513-0196-9 urn:nbn:se:uu:diva-336794 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-336794) For the curious List of Papers This thesis is based on the following papers, which are referred to in the text by their Roman numerals. I Martijn, J*., Lind, AE*., Spiertz, I., Juzokaite, L., Bunikis, I., Petters- son, OV., Ettema TJG. (2018) Amplicon sequencing of the 16S-ITS-23S rRNA operon with long-read technology for improved phylogenetic classification of uncultured prokaryotes. bioRxiv, doi:10.1101/234690 II Lind, AE., Lewis, WH., Spang, A., Guy, L., Embley, TM., Ettema, TJG. (2018) Genomic insights into two independent events of archaeal endo- symbiosis. Manuscript III Lewis, WH*., Lind, AE*., Sendra, KM., Gustafson, HO., Williams, TA., Esteban, GF., Hirt, RP., Ettema, TJG., Embley, TM. (2018) The roles of exaptation, gene loss and gene transfer in the evolution of ciliate hydrogenosomes. Manuscript IV Lind, AE*., Martijn, J*., Schön, ME., Vosseberg, J., Williams, T., Spang, A., Ettema, TJG. (2018) First genomes of Marine Group IV ar- chaea enlighten the evolutionary origins of Haloarchaea. Manuscript * Equal contribution Papers by the author not included in this thesis Baker, BJ., Saw, JH., Lind, AE., Lazar, CS., Hinrichs K., Teske, AP., Ette- ma TJG. (2016) Genomic inference of the metabolism of cosmopolitan sub- surface Archaea, Hadesarchaea. Nature microbiology, 1:16002 Spang A., Saw, JH., Jørgensen, SL., Zaremba-Niedzwiedzka, K., Martijn, J., Lind, AE., van Eijk, R., Schleper, C., Guy, L., Ettema, TJG. (2015) Com- plex archaea that bridge the gap between prokaryotes and eukaryotes. Na- ture, 521(7551):173-179 Schneider, DI., Klasson, L., Lind, AE., Miller, WJ. (2014) More than fish- ing in the dark: PCR of a dispersed sequence produces simple but ultrasensi- tive Wolbachia detection. BMC Microbiology, 14(1):121 Spang, A., Martijn, J., Saw, JH., Lind, AE., Guy, L., Ettema, TJG. (2013) Close encounters of the Third Domain: the emerging genomic view of ar- chaeal diversity and evolution. Archaea, 2013 Bernander, R., Lind, AE., Ettema, TJG. (2011) Archaeal origin of the actin cytoskeleton: implications for eukaryogenesis. Communicative & integrative biology, 4(6):664-667 Contents Introduction ..................................................................................................... 9 The rise of DNA sequencing ......................................................................... 10 Early history ............................................................................................. 10 The high throughput revolution ................................................................ 12 Putting the pieces together: genome assembly ......................................... 15 From micro to pico ................................................................................... 16 The future ................................................................................................. 17 Culture independence .................................................................................... 20 Who is out there? ...................................................................................... 22 What are they doing? ................................................................................ 25 Single cell genomics ................................................................................. 26 Metagenomics ........................................................................................... 27 What we can learn .................................................................................... 29 Problems with culture-independent methods ........................................... 29 Genomics of symbionts ................................................................................. 31 Symbiosis .................................................................................................. 31 Archaeal endosymbionts ........................................................................... 35 Hydrogen-producing mitochondria .......................................................... 40 Evolution of the Haloarchaea ........................................................................ 42 Results ........................................................................................................... 44 Paper I – PacBio sequencing of rRNA amplicons .................................... 44 Paper II – Genome sequences of archaeal endosymbionts ....................... 45 Paper III – Evolution of hydrogenosomes in ciliates ............................... 46 Paper IV –Marine Group IV and the evolution of Haloarchaea ............... 47 Perspectives ................................................................................................... 48 Svensk sammanfattning ................................................................................. 49 Acknowledgements ....................................................................................... 52 References ..................................................................................................... 55 Abbreviations BES 2‐bromoethanesulfonate bp Base pairs CPR Candidate Phyla Radiation ddNTP di-deoxynucleotide triphosphates ETC Electron Transport Chain FISH Fluorescent in situ hybridization HGP Human Genome Project HGT Horizontal Gene Transfer ITS Internal transcribed spacer Kb Kilo base pairs (1,000 bp) KTH Kungliga Tekniska Högskolan (Royal Institute of Technology) LB Lysogeny Broth MAG Metagenome-assembled Genome Mb Megabase (1,000,000 bp) MG-IV Marine Group IV PPi Pyrophosphate SAG Single Amplified Genome SBL Sequence by Ligation SBS Sequence by Synthesis OLC Overlap-Layout-Consensus WGA Whole Genome Amplification Introduction In this thesis I hope to provide a thorough introduction to both the methods used as well as our current understanding of the questions addressed in the included papers. The topics of the papers are wide, but they
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