John Wiley & Sons Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria Cover A guide to state-of-the-art molecular tools for monitoring and managing the toxigenicity of cyanobac.. Product #: 978-1-119-33210-7 Regular price: $139.25 $139.25 In Stock

Molecular Tools for the Detection and Quantification of Toxigenic Cyanobacteria

Kurmayer, Rainer / Sivonen, Kaarina / Wilmotte, Annick / Salmaso, Nico (eds.)

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1. Edition September 2017
440 Pages, Hardcover
Wiley & Sons Ltd
Kurmayer, Rainer / Sivonen, Kaarina / Wilmotte, Annick / Salmaso, Nico (Editor)

ISBN: 978-1-119-33210-7
John Wiley & Sons

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A guide to state-of-the-art molecular tools for monitoring and managing the toxigenicity of cyanobacteria

Runaway eutrophication and climate change has made the monitoring and management of toxigenic organisms in the world's bodies of water more urgent than ever. In order to influence public policy regarding the detection and quantification of those organisms, it is incumbent upon scientists to raise the awareness of policy makers concerning the increased occurrence of toxigenic cyanobacteria and the threats they pose. As molecular methods can handle many samples in short time and help identify toxigenic organisms, they are reliable, cost-effective tools available for tracking toxigenic cyanobacteria worldwide. This volume arms scientists with the tools they need to track toxigenicity in surface waters and food supplies and, hopefully, to develop new techniques for managing the spread of toxic cyanobacteria.

This handbook offers the first comprehensive treatment of molecular tools for monitoring toxigenic cyanobacteria. Growing out of the findings of the landmark European Cooperation in Science and Technology Cyanobacteria project (CYANOCOST), it provides detailed, practical coverage of the full array of available molecular tools and protocols, from water sampling, nucleic acid extraction, and downstream analysis--including PCR and qPCR based methods--to genotyping (DGGE), diagnostic microarrays, and community characterization using next-gen sequencing techniques.
* Offers an overview of the latest trends in the field, while providing a foundation for understanding and applying the tools and techniques described
* Provides detailed coverage of the full range of molecular tools currently available, with expert guidance on the analysis and interpretation of results
* Includes step-by-step guidance on standard operational procedures, including molecular tests used in environmental monitoring, with individual chapters devoted to each procedure
* Complements the published Handbook of Cyanobacterial Monitoring and Cyanotoxin Analysis from the CyanoCOST project

This handbook is an indispensable working resource for scientists, lab technicians, and water management professionals and an excellent text/reference for graduate students and supervisors who use molecular tools. It will also be of great value to environmental health and protection officials and policy makers.

List of Contributors xix

About the Editors xxiii

About the Book xxvii

Preface xxix

Acknowledgments xxxi

1 Introduction 1
Rainer Kurmayer, Kaarina Sivonen, and Nico Salmaso

1.1 A Brief Historical Overview 1

1.2 The Genetic Basis of Toxin Production 2

1.2.1 Microcystin and Nodularin 2

1.2.2 Cylindrospermopsin 5

1.2.3 Saxitoxin 6

1.2.4 Anatoxin 8

1.3 Application of Molecular Tools 8

1.4 Laboratory Safety Issues 13

1.5 References 14

2 Sampling and Metadata 19
Rainer Kurmayer, Guntram Christiansen, Konstantinos Kormas, Wim Vyverman, Elie Verleyen, Vitor Ramos, Vitor Vasconcelos, and Nico Salmaso

2.1 Introduction 19

2.2 Handling of Samples 20

2.3 Sample Contamination 21

2.4 Sampling 21

2.4.1 Quantitative Depth-Integrated and Discrete Sampling 21

2.4.2 Qualitative Plankton Net Sampling 22

2.4.3 Surface (Scum Material) Sampling 22

2.4.4 Benthic (Terrestrial) Cyanobacteria Sampling 22

2.4.5 Food Supplement Sampling 22

2.4.6 Isolation of Single Colonies/Filaments 22

2.5 Subsampling Food Supplement Samples 23

2.6 Sampling of Nucleic Acids 23

2.7 General Conclusions 24

2.8 References 24

SOP 2.1 Sampling and Filtration (DNA) 26
Rainer Kurmayer and Konstantinos Kormas

SOP 2.1.1 Introduction 26

SOP 2.1.2 Experimental 26

SOP 2.1.3 Procedure 27

SOP 2.1.4 Notes 28

SOP 2.1.5 References 29

SOP 2.2 Sampling of Benthic Cyanobacteria 29
Wim Vyverman and Elie Verleyen

SOP 2.2.1 Introduction 29

SOP 2.2.2 Experimental 30

SOP 2.2.3 Procedure 30

SOP 2.2.4 Notes 31

SOP 2.2.5 References 31

SOP 2.3 Isolation of Single Cyanobacteria Colonies/Filaments 32

Rainer Kurmayer

SOP 2.3.1 Introduction 32

SOP 2.3.2 Experimental 32

SOP 2.3.3 Procedure 33

SOP 2.3.4 Notes 33

SOP 2.3.5 References 33

SOP 2.4 Sampling Food Supplements 34
Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos

SOP 2.4.1 Introduction 34

SOP 2.4.2 Experimental 35

SOP 2.4.3 Procedure (Fig. 8.3) 35

SOP 2.4.4 Notes 36

SOP 2.4.5 References 36

SOP 2.5 Sampling and Filtration (RNA) 37
Rainer Kurmayer and Guntram Christiansen

SOP 2.5.1 Introduction 37

SOP 2.5.2 Experimental 37

SOP 2.5.3 Procedure 38

SOP 2.5.4 Notes 38

SOP 2.5.5 References 38

SOP 2.6 Sampling of Abiotic and Biotic Data and Recording Metadata 39
Elie Verleyen, Maxime Sweetlove, Dagmar Obbels, and Wim Vyverman

SOP 2.6.1 Introduction 39

SOP 2.6.2 Experimental 39

SOP 2.6.3 Type of Metadata and Additional Biotic and Abiotic Data 40

SOP 2.6.4 Notes 41

SOP 2.6.5 References 42

3 Isolation, Purification, and Cultivation of Toxigenic Cyanobacteria 43
Sigrid Haande, Iwona Jasser, Muriel Gugger, Camilla H.C. Hagman, Annick Wilmotte, and Andreas Ballot

3.1 Introduction 43

3.2 Methodical Principles for Cyanobacterial Isolation, Purification, and Cultivation 44

3.2.1 Sampling, Identification, and Treatments Prior to the Isolation of Cyanobacteria 44

3.2.2 Traditional Techniques for the Isolation and Purification of Cyanobacteria 45

3.2.3 Culture Media Preparation 47

3.2.4 Cultivation Conditions 48

3.3 General Conclusions 49

3.4 References 49

SOP 3.1 Isolation, Purification, and Clonal Isolate Testing 51
Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot

SOP 3.1.1 Introduction 51

SOP 3.1.2 Experimental 51

SOP 3.1.3 Procedure 52

SOP 3.1.4 Notes 54

SOP 3.1.5 References 54

SOP 3.2 Isolation of Picocyanobacterial Cells by Flow Cytometer (FCM) Sorting 55
Ewa Koz³owska and Iwona Jasser

SOP 3.2.1 Introduction 55

SOP 3.2.2 Experimental 56

SOP 3.2.3 Procedure 56

SOP 3.2.4 Notes 58

SOP 3.2.5 References 59

SOP 3.3 Axenization 60
Muriel Gugger

SOP 3.3.1 Introduction 60

SOP 3.3.2 Experimental 60

SOP 3.3.3 Procedure 61

SOP 3.3.4 Notes 63

SOP 3.3.5 References 63

SOP 3.4 Culture Media (Solid and Liquid) 64
Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot

SOP 3.4.1 Introduction 64

SOP 3.4.2 Experimental 64

SOP 3.4.3 Procedure 65

SOP 3.4.4 Notes 68

SOP 3.4.5 References 68

SOP 3.5 Strain Maintenance (Living Cultures) 69
Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot

SOP 3.5.1 Introduction 69

SOP 3.5.2 Experimental 69

SOP 3.5.3 Procedure 70

SOP 3.5.4 Notes 72

SOP 3.5.5 References 73

SOP 3.6 Cryopreservation and Recovery 73
Muriel Gugger

SOP 3.6.1 Introduction 73

SOP 3.6.2 Experimental 74

SOP 3.6.3 Procedure 75

SOP 3.6.4 Notes 78

SOP 3.6.5 References 78

4 Taxonomic Identification of Cyanobacteria by a Polyphasic Approach 79
Annick Wilmotte, H. Dail Laughinghouse IV, Camilla Capelli, Rosmarie Rippka, and Nico Salmaso

4.1 Introduction 79

4.2 Nomenclature and Classification of Cyanobacteria 82

4.3 Microscopy 84

4.3.1 Light Microscopy 84

4.3.2 Autofluorescence Microscopy 86

4.4 Molecular Markers: Single Loci 87

4.5 Molecular Markers: Multiple Loci 94

4.5.1 Multilocus Sequence Typing (MLST) and Multilocus Sequence Analysis (MLSA) 94

4.5.2 Genome-Based Extension of MLST and MLSA 96

4.6 Molecular Typing Methods Based on Gel Electrophoresis 96

4.7 Denaturing Gradient Gel Electrophoresis (DGGE) 97

4.8 Taxonomic and Molecular Databases 97

4.9 The Polyphasic Approach 98

4.10 Final Considerations 105

4.11 References 106

SOP 4.1 Taxonomic Identification by Light Microscopy 120
Nico Salmaso, Rosmarie Rippka, and Annick Wilmotte

SOP 4.1.1 Introduction 120

SOP 4.1.2 Experimental 121

SOP 4.1.3 References 124

SOP 4.2 Polyphasic Approach on Cyanobacterial Strains 125
Nico Salmaso, Camilla Capelli, Rosmarie Rippka, and Annick Wilmotte

SOP 4.2.1 Introduction 125

SOP 4.2.2 Experimental 126

SOP 4.2.3 References 131

5 Nucleic Acid Extraction 135
Elke Dittmann, Anne Rantala-Ylinen, Vitor Ramos, Vitor Vasconcelos, Guntram Christiansen, and Rainer Kurmayer

5.1 Introduction 135

5.2 Specific Extraction Procedures and Storage 137

5.2.1 DNA Extraction from Laboratory Strains 137

5.2.2 DNA Extraction from Field Samples 137

5.2.3 DNA Extraction from Food Supplements 137

5.2.4 RNA Extraction from Laboratory Strains 138

5.2.5 RNA Extraction from Field Samples 138

5.2.6 Single Colony and Filament Analysis 138

5.2.7 Whole Genome Amplification 139

5.2.8 Nucleic Acid Storage 139

5.3 References 139

SOP 5.1 Standard DNA Isolation Technique for Cyanobacteria 140
Elke Dittmann

SOP 5.1.1 Introduction 140

SOP 5.1.2 Experimental 140

SOP 5.1.3 Procedure 141

SOP 5.1.4 Notes 141

SOP 5.1.5 References 142

SOP 5.2 DNA Isolation Protocol for Cyanobacteria with Extensive Mucilage 143
Guntram Christiansen, Elisabeth Entfellner, and Rainer Kurmayer

SOP 5.2.1 Introduction 143

SOP 5.2.2 Experimental 143

SOP 5.2.3 Procedure 144

SOP 5.2.4 Notes 145

SOP 5.2.5 References 145

SOP 5.3 Quantitative DNA Isolation from Filters 145
Rainer Kurmayer

SOP 5.3.1 Introduction 146

SOP 5.3.2 Experimental 146

SOP 5.3.3 Procedure 147

SOP 5.3.4 Notes 148

SOP 5.3.5 References 148

SOP 5.4 Genomic DNA Extraction from Single Filaments/Colonies for Multiple PCR Analyses 149
Guntram Christiansen, Chen Qin, and Rainer Kurmayer

SOP 5.4.1 Introduction 149

SOP 5.4.2 Experimental 149

SOP 5.4.3 Procedure 150

SOP 5.4.4 Notes 151

SOP 5.4.5 References 151

SOP 5.5 Whole Genome Amplification Using Bacteriophage Phi29 DNA Polymerase 151
Guntram Christiansen and Rainer Kurmayer

SOP 5.5.1 Introduction 151

SOP 5.5.2 Experimental 152

SOP 5.5.3 Procedure 152

SOP 5.5.4 Notes 152

SOP 5.5.5 Reference 153

SOP 5.6 DNA Extraction from Food Supplements 153
Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos

SOP 5.6.1 Introduction 153

SOP 5.6.2 Experimental 153

SOP 5.6.3 Procedure 154

SOP 5.6.4 Notes 155

SOP 5.6.5 References 156

SOP 5.7 RNA Extraction from Cyanobacteria 156
Guntram Christiansen and Rainer Kurmayer

SOP 5.7.1 Introduction 156

SOP 5.7.2 Experimental 156

SOP 5.7.3 Procedure 158

SOP 5.7.4 Notes 158

SOP 5.7.5 References 159

SOP 5.8 cDNA Synthesis 159
Guntram Christiansen and Rainer Kurmayer

SOP 5.8.1 Introduction 159

SOP 5.8.2 Experimental 159

SOP 5.8.3 Procedure 160

SOP 5.8.4 Notes 161

SOP 5.8.5 References 161

6 Conventional PCR 163
Elke Dittmann, Anne Rantala-Ylinen, Kaarina Sivonen, Ilona Ga²ga³a, Joanna Mankiewicz-Boczek, Samuel Cirés, Andreas Ballot, Guntram Christiansen, Rainer Kurmayer, Vitor Ramos, Vitor Vasconcelos, and Martin Saker

6.1 Introduction 163

6.2 Principle of PCR and Available Enzymes 164

6.2.1 Primer Development 165

6.2.2 Setup of PCR Conditions for DNA and Single Colony Analysis 168

6.2.3 Gel Electrophoresis and Documentation 168

6.2.4 Troubleshooting of PCR Results 168

6.2.5 PCR Product Downstream Processing (RFLP, Cloning, Sequencing) 169

6.3 Special Notes 170

6.4 References 170

SOP 6.1 PCR Detection of Microcystin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 172
Elke Dittmann

SOP 6.1.1 Introduction 172

SOP 6.1.2 Experimental 172

SOP 6.1.3 Procedure 173

SOP 6.1.4 Notes 174

SOP 6.1.5 Reference 174

SOP 6.2 PCR Detection of Microcystin and Nodularin Biosynthesis Genes in the Cyanobacterial Orders Oscillatoriales, Chroococcales, Stigonematales, and Nostocales 175
Elke Dittmann, Joanna Mankiewicz-Boczek, and Ilona Ga²ga³a

SOP 6.2.1 Introduction 175

SOP 6.2.2 Experimental 175

SOP 6.2.3 Procedure 177

SOP 6.2.4 Notes 177

SOP 6.2.5 References 178

SOP 6.3 Genus-Specific PCR Detection of Microcystin Biosynthesis Genes in Anabaena/Nodularia and Microcystis and Planktothrix, Respectively 179
Anne Rantala-Ylinen and Kaarina Sivonen

SOP 6.3.1 Introduction 179

SOP 6.3.2 Experimental 179

SOP 6.3.3 Procedure 181

SOP 6.3.4 Notes 181

SOP 6.3.5 References 181

SOP 6.4 PCR Detection of Anatoxin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 182
Anne Rantala-Ylinen and Kaarina Sivonen

SOP 6.4.1 Introduction 182

SOP 6.4.2 Experimental 182

SOP 6.4.3 Procedure 183

SOP 6.4.4 Notes 184

SOP 6.4.5 Reference 184

SOP 6.5 PCR Detection of the Saxitoxin Biosynthesis Genes, sxtA, sxtX, sxtH, sxtG, and sxtI 185
Andreas Ballot and Samuel Cirés

SOP 6.5.1 Introduction 185

SOP 6.5.2 Experimental 187

SOP 6.5.3 Procedure 187

SOP 6.5.4 Notes 188

SOP 6.5.5 References 189

SOP 6.6 PCR Detection of the Cylindrospermopsin Biosynthesis Gene cyrJ 189
Samuel Cirés and Andreas Ballot

SOP 6.6.1 Introduction 189

SOP 6.6.2 Experimental 190

SOP 6.6.3 Procedure 191

SOP 6.6.4 Notes 191

SOP 6.6.5 References 192

SOP 6.7 PCR from Single Filament of Toxigenic Planktothrix 193
Qin Chen, Guntram Christiansen, and Rainer Kurmayer

SOP 6.7.1 Introduction 193

SOP 6.7.2 Experimental 193

SOP 6.7.3 Procedure 194

SOP 6.7.4 Notes 195

SOP 6.7.5 References 195

SOP 6.8 Analysis of Microcystin Biosynthesis Gene Subpopulation Variability in Planktothrix 196
Rainer Kurmayer

SOP 6.8.1 Introduction 196

SOP 6.8.2 Experimental 196

SOP 6.8.3 Procedure 197

SOP 6.8.4 Notes 197

SOP 6.8.5 References 198

SOP 6.9 PCR Detection of Microcystin Biosynthesis Genes from Food

Supplements 199
Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos

SOP 6.9.1 Introduction 199

SOP 6.9.2 Experimental 199

SOP 6.9.3 Procedure 201

SOP 6.9.4 Notes 202

SOP 6.9.5 References 203

7 Quantitative PCR 205
Anne Rantala-Ylinen, Henna Savela, Kaarina Sivonen, and Rainer Kurmayer

7.1 Introduction 205

7.2 Primer/Probe Design 206

7.3 Optimization 208

7.4 Absolute Quantification 208

7.5 Relative Quantification 209

7.6 Calibration of qPCR Results 209

7.7 General Conclusions 210

7.8 References 210

SOP 7.1 Optimization of qPCR Assays 211
Rainer Kurmayer

SOP 7.1.1 Introduction 211

SOP 7.1.2 Experimental 211

SOP 7.1.3 Procedure 212

SOP 7.1.4 Notes 213

SOP 7.1.5 References 213

SOP 7.2 Calibration of qPCR Results 214
Rainer Kurmayer

SOP 7.2.1 Introduction 214

SOP 7.2.2 Experimental 214

SOP 7.2.3 Procedure 215

SOP 7.2.4 Notes 217

SOP 7.2.5 References 217

SOP 7.3 Quantification of Potentially Microcystin/Nodularin-Producing Anabaena, Microcystis, Planktothrix, and Nodularia 218
Anne Rantala-Ylinen, Kaarina Sivonen, and Rainer Kurmayer

SOP 7.3.1 Introduction 218

SOP 7.3.2 Experimental 219

SOP 7.3.3 Procedure 219

SOP 7.3.4 Notes 221

SOP 7.3.5 References 221

SOP 7.4 Relative Quantification of Microcystis or Planktothrix mcy Genotypes Using qPCR 222
Rainer Kurmayer

SOP 7.4.1 Introduction 222

SOP 7.4.2 Experimental 222

SOP 7.4.3 Procedure 224

SOP 7.4.4 Notes 225

SOP 7.4.5 References 225

SOP 7.5 Quantification of Transcript Amounts of mcy Genes in Planktothrix 226
Guntram Christiansen and Rainer Kurmayer

SOP 7.5.1 Introduction 226

SOP 7.5.2 Experimental 226

SOP 7.5.3 Procedure 227

SOP 7.5.4 Notes 228

SOP 7.5.5 References 228

SOP 7.6 Quantification of Potentially Cylindrospermopsin-Producing Chrysosporum ovalisporum 229
Rehab El-Shehawy and Antonio Quesada

SOP 7.6.1 Introduction 229

SOP 7.6.2 Experimental 229

SOP 7.6.3 Procedure 230

SOP 7.6.4 Notes 231

SOP 7.6.5 References 231

SOP 7.7 qPCR Detection of the Paralytic Shellfish Toxin Biosynthesis Gene sxtB 231
Henna Savela

SOP 7.7.1 Introduction 231

SOP 7.7.2 Experimental 232

SOP 7.7.3 Procedure 233

SOP 7.7.4 Notes 234

SOP 7.7.5 References 234

SOP 7.8 Application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines to Quantitative Analysis of Toxic Cyanobacteria 234
Henna Savela

SOP 7.8.1 Introduction 234

SOP 7.8.2 Sampling 235

SOP 7.8.3 Sample Preparation and DNA Extraction 235

SOP 7.8.4 Target Information and Oligonucleotide Design 235

SOP 7.8.5 qPCR Protocol 238

SOP 7.8.6 qPCR Validation 239

SOP 7.8.7 Data Analysis 239

SOP 7.8.8 Reference 239

8 DNA (Diagnostic) and cDNA Microarray 241
Anne Rantala-Ylinen, Kaarina Sivonen, and Annick Wilmotte

8.1 DNA (Diagnostic) Microarray 241

8.1.1 Introduction 241

8.1.2 Methodological Principles 242

8.1.3 General Conclusions 243

8.1.4 References 243

8.2 cDNA Microarray for Cyanobacteria 244
Hans C.P. Matthijs and J. Merijn Schuurmans

8.2.1 Introduction 244

8.2.2 Principles of Microarray Use 244

8.2.3 Considerations for Experimental Design 245

8.2.4 Microarray: Practical Approach 246

8.2.5 Microarray: Data Analysis 246

8.2.6 References 246

SOP 8.1 DNA-Chip Detection of Potential Microcystin and Nodularin Producing Cyanobacteria in Environmental Water Samples 248
Anne Rantala-Ylinen and Kaarina Sivonen

SOP 8.1.1 Introduction 248

SOP 8.1.2 Experimental 249

SOP 8.1.3 Procedure 250

SOP 8.1.4 Notes 253

SOP 8.1.5 References 253

SOP 8.2 cDNA Microarrays for Cyanobacteria 254
J. Merijn Schuurmans and Hans C.P. Matthijs

SOP 8.2.1 Introduction 254

SOP 8.2.2 Experimental 254

SOP 8.2.3 Procedure 256

SOP 8.2.4 Notes 259

SOP 8.2.5 Reference 261

9 Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis 263
Iwona Jasser, Aleksandra Bukowska, Jean-Francois Humbert, Kaisa Haukka, and David P. Fewer

9.1 Introduction 263

9.2 Main Applications of the Method 264

9.3 Possible Applications 264

9.4 DGGE Procedure 265

9.5 General Conclusions Including Pros and Cons of the Method 267

9.6 Optimization of the Method and Troubleshooting 267

9.7 References 268

SOP 9.1 DGGE-mcyA Conditions 270
Aleksandra Bukowska and Iwona Jasser

SOP 9.1.1 Introduction 270

SOP 9.1.2 Experimental 270

SOP 9.1.3 Procedure 272

SOP 9.1.4 Notes 275

SOP 9.1.5 References 275

10 Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques 277
Li Deng, Maxime Sweetlove, Stephan Blank, Dagmar Obbels, Elie Verleyen, Wim Vyverman, and Rainer Kurmayer

10.1 Introduction 277

10.2 Specific Procedures 279

10.2.1 16S rRNA Gene Amplicon Library Preparation 279

10.2.2 Amplicon Purification, Quantification and Pooling 280

10.2.3 Sequencing 280

10.2.4 Bioinformatic Exploration of Sequencing Results 281

10.2.5 General Conclusions Including Pros and Cons of the Method 281

10.2.6 References 281

10.3 Bioinformatic Processing of Amplicon Sequencing Datasets 283
Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman

10.3.1 Introduction 283

10.3.2 Sequencing Platforms 283

10.3.3 Data Formats 284

10.3.4 Error Associated with NGS Data 285

10.3.5 OTU Delineation: Choosing a Similarity Threshold 286

10.3.6 Conclusions 286

10.4 References 286

SOP 10.1 Standard Technique to Generating 16S rRNA PCR Amplicons for NGS 288
Li Deng, Stephan Blank, Guntram Christiansen, and Rainer Kurmayer

SOP 10.1.1 Introduction 288

SOP 10.1.2 Experimental 288

SOP 10.1.3 Procedure 289

SOP 10.1.4 Notes 290

SOP 10.1.5 References 290

SOP 10.2 Bioinformatics Analysis for NGS Amplicon Sequencing 291
Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman

SOP 10.2.1 Introduction 291

SOP 10.2.2 Experimental 291

SOP 10.2.3 Practical Tips and Alternatives for Quality Filtering 298

SOP 10.2.4 References 298

11 Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production 301
Vitor Ramos, Cristiana Moreira, Joanna Mankiewicz-Boczek, and Vitor Vasconcelos

11.1 Introduction 301

11.2 Possible Applications 303

11.3 Checklist of Publications, Applications and Lessons from Practice 315

11.3.1 Molecular-Based Studies on (Toxic) Cyanobacteria: Overview of Methods Being Used, and Generic Findings and Concerns 315

11.3.2 The Need for Complementary Approaches 316

11.3.3 Interpreting Results 316

11.3.4 Choice of Molecular Tools for Toxigenicity Assessment 317

11.3.5 Common and Possible Applications of Molecular Tools 318

11.4 General Conclusions 321

11.5 Acknowledgments 324

11.6 References 324

Appendix: Supplementary Tables 335

Cyanobacterial Species Cited in the Book 376

Glossary 379

Index 393
Rainer Kurmayer, PhD, is an associate professor and Head of the Univ. of Innsbruck Research Institute of Limnology, Innsbruck, Austria.

Kaarina Sivonen, PhD, is aprofessor of microbiology at the University of Helsinki, Finland.

Annick Wilmotte is a research associate and senior scientist at the FRS-FNRS, University of Liège, Belgium.

Nico Salmaso, PhD, isHead of the Hydrobiology Unit of the Istituto Agrario di S. Michele All'Adige, Fondazione E. Mach (FEM), Trentino, Italy.