Introduction to Marine Genomics (eBook)

Preface 6
Contents 10
Contributors 12
1 Genomics in the Discovery and Monitoring of Marine Biodiversity 16
1.1 Marine Biodiversity and Genomics A Global Perspective 16
1.1.1 Marine Biodiversity: Structural and Functional Components 16
1.1.2 The Nature of Marine Biodiversity 21
1.1.3 Empirical and Conceptual Advances 21
1.2 Molecular Identification of Marine Biodiversity 23
1.2.1 Diversity and Functional Analyses of Microbial Communities 25
1.2.2 Between the Microbes and Metazoans: Eukaryotic Protists 27
1.2.2.1 Ribosomal Probes 27
1.2.2.2 Biodiversity Assessment at Sub-species Level 28
1.2.3 Diversity and Ecological Analyses of Benthic Meiofaunal Communities 29
1.2.4 DNA Barcoding and Fisheries 30
1.2.5 Larvae in Marine Systems 32
1.3 Marine Biodiversity and Ecosystem Function 36
1.3.1 Microbes in Novel Environments 36
1.3.2 Microbial Links in Ecosystem Processes 36
1.3.3 Environmental Change and Microbial Diversity 37
1.4 Concluding Remarks 38
References 40
2 Metagenome Analysis 48
2.1 Introduction 48
2.2 History and Application of Metagenomics 50
2.3 Technical Challenges in Metagenome Analysis 52
2.3.1 Strategies to Assess the Metagenome 52
2.3.2 Enrichment Strategies 54
2.3.3 Isolation and Purification of Genomic DNA 56
2.3.4 Amplification of Genomic DNA 57
2.3.5 Construction and Analysis of Metagenomic Libraries 60
2.3.5.1 Small Insert Metagenomic Libraries 60
2.3.5.2 Large Insert Metagenomic Libraries 60
2.3.5.3 Metagenomic Library Size 62
2.3.5.4 Storage of Metagenomic Libraries 63
2.3.5.5 Screening of Metagenomic Libraries 64
2.3.6 Library Independent Metagenome Analysis 64
2.4 Bioinformatic Challenges in Metagenome Analysis 66
2.4.1 Fragment Assembly assembly and Binning 67
2.4.2 Gene Prediction 69
2.4.3 Functional Annotation 69
2.4.4 Web Based Annotation Pipelines 70
2.4.5 Annotation Systems for Local Installation 71
2.4.6 High Diversity Environments, Shallow Sequencing and Short Read Technologies 72
2.4.7 Metagenome Descriptors for Comparative Metagenomics 73
2.4.7.1 Phylogenetic Diversity 73
2.4.7.2 Functional Diversity 74
2.5 Outlook 75
References 77
3 Populations and Pathways: Genomic Approachesto Understanding Population Structure and EnvironmentalAdaptation 87
3.1 Tools 88
3.1.1 DNA and RNA Studies: EST Libraries 89
3.1.2 DNA Studies: Microsatellites 90
3.1.3 DNA Studies: Single Nucleotide Polymorphisms (SNPs) 91
3.1.4 DNA Studies: Amplified Fragment Length Polymorphisms (AFLPs) 92
3.1.5 DNA Studies: High Through-Put Sequencing 94
3.1.6 DNA and RNA Studies: Targeted Gene Analyses 95
3.1.7 DNA Studies: Barcoding 96
3.1.8 RNA Studies: Microarrays or Gene Chips 96
3.1.9 RNA Studies: Q-PCR 97
3.2 Population Genomics 97
3.2.1 Analysis: Choices, Limitations and Considerations 98
3.2.1.1 Marker Type 98
3.2.1.2 Differentiating Selective and Demographic Effects 100
3.2.1.3 Identifying Adaptive Traits 100
3.3 Practical Application of Population Genomics in the Marine Environment 104
3.3.1 Dispersal in the Sea: From Larval Development to Local Adaptation and Speciation Processes 104
3.3.1.1 Pelagic Larval Studies 104
3.3.1.2 Genetic Basis of Adaptive Differentiation in High Gene Flow Species 105
3.3.1.3 Study of Hybrid Zones and the Speciation Processes 106
3.3.2 Marine Bio-Invasions: Using Genomic Resources to Study Invasive Species 107
3.3.3 Uncovering the Genetic Basis of Hybrid Vigour in Aquaculture Populations 107
3.3.4 Gene Polymorphism and Population Adaptation 109
3.4 Expression Studies and Environmental Genomics 110
3.4.1 Defining Habitat Limits: Biogeography 111
3.4.2 Microarrays: Identification of Biochemical Pathways Involved in Adaptation 113
3.4.3 Genome Plasticity and Seasonal Variation 113
3.4.4 Adaptation to Extreme Environments 114
3.4.4.1 Hydrothermal Vents 115
3.4.4.2 Polar Environments 116
3.4.4.3 Ecotoxicology Monitoring 118
3.5 Summary and Future Issues 119
References 120
4 Phylogeny of Animals: Genomes Have a Lot to Say 133
4.1 Introduction 133
4.2 The Roots of Animal Phylogeny 135
4.2.1 Historical Schemes Are Based on the Coelom Evolution Hypotheses 135
4.2.2 Sorting More Characters Through a Cladistic Approach 136
4.2.3 Small Ribosomal RNA Gene and the ''New View'' of Animal Phylogeny 139
4.2.4 The Limits of the ''New View'' 140
4.3 The Power and Pitfalls of Phylogenomics 141
4.4 Phylogenomics Resolves Animal Relationships 142
4.4.1 Battle over the Coelomata and the Importance of Taxonomic Sampling 143
4.4.1.1 Early Phylogenomic Attempts Challenged the ''New View'' 143
4.4.1.2 Coelomata and the Interpretation of Rare Genomic Changes 143
4.4.2 Is It Actually Possible to Decipher Animal Relationships? 145
4.5 Toward a Broad Phylogenomic Picture of Metazoan Relationships 146
4.5.1 Challenging Well-Established Clades: The Case of Deuterostomes 146
4.5.2 Chaetognaths Fit into the Bilaterian Tree 148
4.5.3 Acoel Flatworms, Basal or Not? 148
4.5.4 Deeper into Protostome Relationships 149
4.6 Conclusion: The Future of Animal Phylogeny 149
References 151
5 Metazoan Complexity 156
5.1 Approaches to Complexity 156
5.2 Choanoflagellates: The Evolution of Multicellularity in Metazoa 159
5.3 Sponges: The Evolution of Animal Development, Body Axis, Cell Types and Epithelia 163
5.4 The Placozoan Trichoplax: A Primitively Simple or Highly Reduced Metazoan? 165
5.5 Cnidaria: A Simple Body with a Complex Genome 167
5.5.1 The Nematostella Genome 168
5.5.2 Cnidarian BMP Patterning and the Evolution of the Bilaterian Dorso-Ventral Axis 169
5.5.3 Cnidarian Hox Genes and the Evolution of the Antero-Posterior Axis 169
5.5.4 The Homology of Body Axes Between Cnidaria and Bilateria 170
5.5.5 Cnidarians and the Evolution of Mesoderm 172
5.5.6 ''Cryptic'' Complexity in Cnidarians? 173
5.6 Ecdysozoans: Going Beyond the Established Systems 174
5.7 Lophotrochozoans: An Evolutionary Branch Leading to New Perspectives 175
5.8 Aplysia: From Neural Circuits to Neurotranscriptomics 175
5.9 Platynereis: Ancestral Complexity of Cells and Genomic Features 176
5.10 Alternative Splicing: Modulating the Basic Layers of Genomic Complexity? 178
5.11 Sea Urchins: Unexpected Functional Repertoires at the Base of Deuterostomes 179
5.12 Lancelets and the Chordate Prototype 180
5.13 Ascidians: Changes and Constants in Developmental Programmes 181
5.14 Perspectives 182
References 184
6 Genomics of Marine Algae 192
6.1 What Are Algae? 192
6.2 Why Algae Are Interesting 193
6.3 Endosymbiosis and the Origins of the Algae 194
6.4 Algae and Marine Ecosystems 196
6.4.1 Diversification of the Phytoplankton During the Evolution of the Earth 200
6.4.2 Algae Are Important Components of the Phytoplankton 200
6.4.3 Exploration of Planktonic Ecosystems Using High-Throughput Sequencing 201
6.4.4 Diversity and Dynamics of Planktonic Ecosystems 203
6.4.5 Organism-Based Approaches for Exploring the Biology of Planktonic Algae 204
6.4.5.1 Diatom Genomics 205
6.4.5.2 Prasinophyte Genomics 208
6.4.5.3 Other Microalgal Genome Projects 210
6.4.5.4 Dinoflagellates 211
6.4.6 Macroalgal Genomics 212
6.4.6.1 Brown Macroalgae 212
6.4.6.2 Red Macroalgae 214
6.4.6.3 Green Macroalgae 216
6.5 Future Research in Algal Genomics 216
References 217
7 Genomic Approaches in Aquaculture and Fisheries 225
7.1 Introduction 225
7.2 Genomic Tools and Resources 227
7.2.1 Genetic Linkage Maps 227
7.2.2 Radiation Hybrid (RH) Maps 230
7.2.3 BAC-Based Physical Maps 231
7.2.4 High Quality Draft Genome Sequences 232
7.2.5 Functional Genomic Tools 232
7.3 Genomic Approaches in Breeding and Reproduction 234
7.4 Genomic Approaches in Growth and Nutrition 237
7.4.1 Introduction 238
7.4.2 Transcriptomic Changes in Skeletal Muscle Related to Muscle Growth 238
7.4.3 Transcriptomic Changes in Skeletal Muscle Related to External Factors 239
7.4.4 Genomic Approaches to the Study of Hepatic Function 240
7.4.4.1 Transcriptional Changes in the Liver in Relation to Growth and Nutrition 241
7.4.4.2 Changes in the Liver Proteome in Relation to Nutrition and Growth 242
7.4.5 Conclusions and Future Directions 243
7.5 Genomic Approaches in Product Quality and Safety 243
7.5.1 Seafood Quality Has a Multifactorial Background 244
7.5.2 Fish Quality Traits Assessed by Genomic and Proteomic Methods 244
7.5.2.1 Colour 245
7.5.2.2 Texture (as Muscle Cellularity) 245
7.5.2.3 Texture (as Affected by Postmortem Degradation) 246
7.5.2.4 Nutritional Quality and Health Value 247
7.5.3 Other Emerging Quality Traits 248
7.5.4 Seafood Safety 249
7.5.4.1 Health Hazards in Seafood 250
7.5.4.2 Allergenicity in Seafood Products 250
7.5.5 Seafood Authentication and Traceability 251
7.6 Genomic Approaches in HostPathogen Interaction 252
7.6.1 Host--Parasite Interactions in Fish 252
7.6.2 Transcriptomic Characterization of Host Immune Response 253
7.6.2.1 EST Analysis to Identify Genes Involved in Host Immune Response 253
7.6.2.2 Microarray Analysis to Identify Genes Involved in Host Immune Response 253
7.6.2.3 Real-Time PCR to Identify Candidate Markers for Disease Detection 254
7.6.3 How Can Genetic Linkage, RH and Physical Maps Contribute to Shedding Light on Fish--Pathogen Interactions? 254
7.6.4 Host--Parasite Interactions in Shellfish 255
7.6.4.1 Improvement of Diagnostic Tools Using Molecular Approaches 255
7.6.4.2 Molecular Immunity of Bivalves 256
7.6.4.3 Immune Response to Perkinsus Infection 257
7.6.4.4 Immune Response to Vibrio Infection 259
7.6.4.5 Status of Transcriptomic Tools 260
7.6.4.6 Conclusions 261
7.7 Genomic Variation, Stock Structure, Adaptation and Traceability in Natural Fish Populations 261
7.7.1 The Major Issues 261
7.7.2 State-of the Art in the Population Genomics of Fishes 264
7.7.2.1 Identifying Population Structure and Dynamics 264
7.7.2.2 Selection and Adaptation in Natural and Exploited Populations 267
7.7.2.3 Tracing Natural Populations for Fisheries Enforcement and Traceability 270
7.7.2.4 Integrating Evolutionary and Ecological Functional Genomics with the Environment 272
7.7.3 A Vision of the Future 274
References 276
8 Marine Biotechnology 299
8.1 A Brief Description of the Field of Marine Biotechnology 299
8.2 How Genomics Impacts on the Various Fields of Marine Biotechnology 301
8.3 Expanding Gene Resources Through Microbial-Community Genomic Projects, Complete Genomes of Isolated Organisms and Data Mining 302
8.3.1 Complete Genomes 303
8.3.2 The Growing Contribution of Metagenomes 305
8.4 Contribution of Marine Biotechnology to the Discovery of Natural Products, Novel Pharmaceuticals and White Technology 311
8.4.1 Viruses 311
8.4.2 Archaea and Bacteria 313
8.4.3 Algae 313
8.4.4 Algae for Biodiesel Production 314
8.4.5 Algae for Ethanol Production 315
8.4.6 Algae for Hydrogen Gas Production 315
8.4.7 Algae for Biomass Fermentation 315
8.4.8 Marine Genomics and Algal Biofuels 316
8.4.9 Algae as a Cell Factory 316
8.4.10 Marine Fungi 318
8.4.11 Metazoans 318
8.4.12 In Closing 319
References 319
9 Practical Guide: Genomic Techniques and How to Apply Them to Marine Questions 326
9.1 Sequence Data Generation 327
9.1.1 Classical Genome Sequencing Approaches 327
9.1.1.1 The Sanger Method 327
9.1.1.2 Shotgun Technique 328
9.1.1.3 Bacterial Genome Assembly and Finishing 328
9.1.2 Next Generation of Genome Sequencing 329
9.1.2.1 Pyrosequencing or 454 Sequencing 331
9.1.2.2 Illumina 0 Sequencing Technology 331
9.1.2.3 SOLiD 0 System 332
9.1.3 Other New Advanced Approaches to DNA Sequencing 332
9.1.3.1 Open-Source ''Polony Sequencing'' System 332
9.1.3.2 Sequencing-by-Hybridization 333
9.1.3.3 Nanopore Sequencing 333
9.1.4 Conclusion 333
9.2 Data Management for Bioinformatics Applications 334
9.2.1 Data Modelling and Storage 334
9.2.2 Data Access 335
9.2.3 Common File Formats 337
9.3 DNA Sequence Analysis 337
9.3.1 EST Processing 337
9.3.2 Gene Prediction 342
9.3.2.1 Gene Finding in Prokaryotes 342
9.3.2.2 Gene Finding in Eukaryotes 344
9.3.3 Genome Annotation and Beyond 348
9.3.3.1 Introduction to Sequence Similarity 348
9.3.3.2 From Gene Annotation to Genome Annotation 349
9.3.3.3 Protein Annotation Tools 350
9.3.4 Comparative Genomics and Functional Classification 353
9.3.4.1 Homology and Similarity 353
9.3.4.2 Protein Domains 355
9.3.4.3 Use of Gene Clusters in Functional Annotation 355
9.3.4.4 Existing Resources for Comparative Analyses 356
9.3.5 Major Public Sequences Databases and Other Resources 358
9.3.5.1 Major Public Nucleotide Sequences Databases 358
9.3.5.2 Major Public Protein Sequences Database: UniProt 362
9.3.5.3 RefSeq 364
9.3.5.4 Other Resources 365
9.4 Transcriptome Analysis Using High-Throughput Technology 367
9.4.1 Fundamentals of Microarray Technology 370
9.4.1.1 Variation and Replication 371
9.4.1.2 How Many Replicates? 372
9.4.2 Gene Expression Analysis 372
9.4.2.1 Image Analysis 372
9.4.2.2 Normalization 373
9.4.2.3 Detecting Significant Changes 374
9.4.2.4 Cluster Analysis 376
9.4.2.5 Classification 376
9.4.2.6 Microarray Software 377
9.4.2.7 Image Analysis Software 377
9.4.2.8 Pure Analysis Systems 378
9.4.2.9 General Purpose Database Systems 378
9.4.2.10 R and BioConductor 378
9.4.3 Data Sharing and Public Repositories 379
9.4.4 Summary of the Gene Expression Analysis Section 380
References 381
Glossary 390
Index 400

"Chapter 2 Metagenome Analysis (p. 33-34)

Anke Meyerdierks and Frank Oliver Glöckner

Abstract The term “metagenomics” represents a combination of molecular and bioinformatic tools used to assess the genetic information of a community without prior cultivation of the individual species. It is valuable for the study of microorganisms of which only a minor fraction is yet culturable. The collective genomes present in an environmental sample or in an enrichment of target cells are extracted and subject to sequence-based or functional analyses.

The field of metagenomics is evolving very rapidly, especially due to newly developed high-throughput sequencing technologies and increased computational power. Metatranscriptome and – proteome analyses are increasingly combined with metagenomic studies in order to assess not only the genetic potential of a microbial community, but also the genes expressed in a particular environment. The present chapter gives a short historical overview of the early years of metagenome analyses, and of possible applications. Challenges regarding the molecular and bioinformatic part of metagenome analyses are discussed.

The molecular section includes strategies to access a metagenome. Methods to enrich for cells or the DNA of certain subpopulations prior to metagenome analysis, as well as to extract, purify and amplify DNA are given. The construction and sequence-based screening of small and large insert metagenomic libraries as well as a library-independent metagenomic approach using a new high throughput sequencing technology are described. The bioinformatic section provides an overview of assembly and binning tools, gene prediction programs, and annotation systems.

This section also addresses the problem of metagenomic studies on habitats with high microbial diversity. Moreover, approaches to analyse phylogenetic and functional diversity within a dataset are discussed. The aim of the chapter is to provide the reader with basic information on both the molecular and bioinformatic aspects of metagenome analysis, to give hints to further reading and, therefore, to enable the reader to use this valuable method in an appropriate way in his or her studies.

2.1 Introduction

Microorganisms are the most abundant form of life on Earth, and catalyse key processes such as nitrogen fixation and the mineralization of organic matter. Considering that about 70% of our Earth’s surface is covered by the oceans, a comprehensive understanding of microbial element cycling in marine environments is crucial if we are to understand these processes on a global scale. In the light of the current discussions about global warming, the formation, storage, emission, as well as degradation of greenhouse gases in marine environments is receiving a lot of attention.

Although the importance of an investigation of biogeochemical cycles in the ocean has generally been acknowledged, the study of microbial populations in marine environments, their contribution to biogeochemical cycles, and the ecophysiology of individual species is still in its infancy. Metagenomics, defined as the cultivation independent approach to assess the genetic potential of organisms, has opened a new dimension in environmental research. This chapter is intended to provide an overview of the recent developments in metagenomics, ranging from lab technology to bioinformatics. Antoni van Leeuwenhoek provided the first microscopic evidence for the existence of microorganisms in the late seventeenth century (for review: Hall 1989)."