This electronic thesis or dissertation has been downloaded from Explore Bristol Research, http://research-information.bristol.ac.uk Author: Nash, Maisie V Title: Metagenomic insights into microbial communities in proglacial landscapes General rights Access to the thesis is subject to the Creative Commons Attribution - NonCommercial-No Derivatives 4.0 International Public License. A copy of this may be found at https://creativecommons.org/licenses/by-nc-nd/4.0/legalcode This license sets out your rights and the restrictions that apply to your access to the thesis so it is important you read this before proceeding. Take down policy Some pages of this thesis may have been removed for copyright restrictions prior to having it been deposited in Explore Bristol Research. 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Metagenomic insights into microbial communities in proglacial landscapes Maisie Victoria Nash A thesis submitted to the University of Bristol in accordance with the requirements for award of the degree of Doctor of Philosophy in the Faculty of Science School of Geographical Sciences April 2019 Word count: 46,231 Abstract Environmental DNA analysis using metagenomics can provide an insight into the taxonomy and functional potential of microbial communities ex situ, without the need for culturing or DNA amplification. However, metagenomics has had limited application to environmental microbial ecology, in particular, to microbial communities in proglacial regions. This thesis aims to contribute to the body of literature on environmental metagenomics through evaluating assemblers for soil microbial ecologists, and subsequently applying metagenomics to investigate microbial communities in proglacial environments. Assembly of metagenome sequencing reads can improve sequence alignment to taxonomic and functional databases, thereby improving ecological conclusions. However, limited guidance is available for assembler choice by microbial ecologists. The first study in this thesis compares assemblers for soil metagenome data, demonstrating the importance of assembler evaluation and parameterization. The guidance produced was applied to investigate microbial communities in proglacial regions, including fjords and forefields. Proglacial forefields present a unique opportunity to understand microbial colonization in land exposed by glacier retreat. Here, metagenomics was used to investigate microbial diversity and functional potential during forefield succession, alongside comparing the diversity of nitrogen-fixing bacteria between Arctic forefields. This work contributes to our understanding of Arctic microbial ecology, which has significance given the continued exposure of forefield soils during global warming. In addition, metagenomics was used to investigate microbial communities in oligotrophic, dark, saline fjord waters, fed by glacial meltwater. This work highlights the potential of metagenomics to understand uncultured microbial samples and demonstrate areas for further analysis, such as targeting novel genomes. This thesis has contributed to the literature on metagenomics by providing methodological guidance for microbial ecologists, alongside enhancing understanding of microbial diversity in proglacial regions. It is hoped that this work will inspire others to use metagenomics to explore uncultured microbial samples and to target further analysis or exploration for unique genomes. 2 Acknowledgements I would like to thank the following people who have provided guidance and support over the course of my PhD. Firstly, I would like to thank my advisors, Patricia Sánchez-Baracaldo who has been a fantastic collaborator and a huge source of support, Alex Anesio for providing me with the opportunity to enter the world of microbiology, and Gary Barker for pushing me to become a better bioinformatician. Thank you to NERC for funding my project through a GW4+ DTP scholarship and the PISCES project for supporting my fieldwork. Many thanks to my cohort of PhD students and the Browns community, in particular Nathan Christmas for being my technical help desk, and Steve Chuter, Ale Urra and Claire Donnelly for supporting me through the ups and downs. I would like to thank my fieldwork team, including Jon Hawkings, Rebecca Huggett, Rory Burford, Alex Beaton, Helena Pryer, Hong Chin and all those on the PISCES project. Thank you to my team and coaches at CrossFit 605 who have kept my competitive spirit alive and motivated me to improve in all aspects of my life. I would like to thank my best friend, Alice Haworth and my sisters, Amy and Libby, for supporting me through my crazy pursuit in science. Finally, I would like to thank my parents, Trevor and Julie, without their support I would never have made it this far, and their work ethic and positive attitude inspires me every day. 3 Authors declaration I declare that the work was carried out in accordance with the requirements of the University’s regulations and Code of Practice for Research Degree programmes and that it has not been submitted for any other academic award. Except where indicated by specific reference in the text, the work is the candidate’s own work. Work done in collaboration with, or the assistance of others, is indicated as such. Any views expressed in the dissertation are those of the author. Signed: ………………………………………………………………. Date:………………… 4 Table of Contents Abstract……………………………………………………………………………………….2 Acknowledgements………………………………………………………………………….3 Authors declaration………………………………………………………………………….4 List of Figures………………………………………………………………………………...9 List of Tables……………………………………………………………………………..…10 Key terms……………………………………………………………………………………11 Abbreviations………………………………………………………………………….……12 Chapter 1: Literature Review……………………………………………………………13 1.1 Introduction to glacial systems………………………………………………..13 1.2 Microbial communities in glacial systems……………………………………14 1.3 Proglacial forefields and microbial communities……………………………14 1.3.1 Using forefields to investigate microbial succession……………15 1.3.2 Plant succession and soil development………………………….16 1.3.3 Microbial succession in glacial forefields…………………………17 1.3.4 Microbial nitrogen cycling in glacial forefields……………………19 1.3.5 Carbon, phosphorous and sulfur in forefields……………………20 1.4 Proglacial fjords and microbial communities………………………………...22 1.4.1 Fjord systems……………………………………………………….22 1.4.2 The influence of glacial meltwater……………………...…………22 1.4.3 Microbial communities and fjord systems……………………..…23 1.5 Proglacial environments and climate change……………………………….24 1.6 Methodological considerations for investigating microbial communities…25 1.6.1 Methods available………………………………………………….25 1.6.2 Culture based studies……………………………………………...25 1.6.3 First generation DNA sequencing………………………………...26 1.6.4 Taxonomic marker genes…………………………….…………...27 1.6.5 Next generation DNA sequencing………………………………...27 1.6.6 Metagenomics……………………………………………………...28 Metagenome sequence assembly and annotation……………...29 Sequence assembly……………………………………………….29 Sequence annotation………………………………………………31 5 The choice of assembler…………………………………………..32 Selecting an appropriate assembler……………………………...33 1.7 Summary and research gaps…………………………………………………34 1.8 Aims and objectives……………………………………………………………35 Chapter 2: Comparison of publicly available metagenome assemblers for soil bacterial communities…………………………………………………………………...38 2.1 Introduction……………………………………………………………………..38 2.2 Methodology……………………………………………………………………40 2.3 Results……………………………………………………………………….…46 2.3.1 Assembly contiguity and completeness……………………………46 2.3.2 Modifying community complexity……………………………….….51 2.3.3 Assembly parameterization…………………………………………52 2.3.4 Assembly chimeras………………………………………………….56 2.3.5 Taxonomic distribution of assemblies……………………………...57 2.4 Discussion………………………………………………………………………59 2.4.1 The premise of the study……………………………………………59 2.4.2 Assembly evaluation metrics……………………………………….59 2.4.3 Simulated bacterial communities…………………………………..60 2.4.4 The choice of assembler………………………………………….…61 2.4.5 The importance of parameterisation……………………………….62 2.4.6 Best performing assemblers………………………………………..62 2.5 Conclusion……………………………………………………………………...63 2.6 Observations……………………………………………………...……………64 2.7 Recommendations for best practice…………………………………………65 2.8 Limitations and future work……………………………………………………66 Chapter 3: Metagenomic insights into diazotrophic communities across Arctic glacier forefields………………………………………………………………………….68 3.1 Introduction……………………………………………………………………..69 3.2 Materials and methods…………………………………………………...……71 3.2.1 Field sampling …………………………………………………….…71 3.2.2 Soil organic carbon and total nitrogen content……………….……72 3.2.3 DNA extraction, library preparation and sequencing…………..…72 3.2.4 Metagenome assembly and annotation……………………………73 3.2.5 Nif taxonomy…………………………………………………………74 6 3.2.6 Gene phylogeny…………………………………………………..…74 3.3 Results and Discussion……………………………………………………..…76 3.3.1 Soil carbon