Microbial Metagenomics, Metatranscriptomics, and Metaproteomics

Series Page

METHODS IN ENZYMOLOGY

Contributors

Preface

Methods in Enzymology

Section I: Preparation and Genomic Analyses of Single Cells from Natural Populations

Chapter One. In-Solution Fluorescence In Situ Hybridization and Fluorescence-Activated Cell Sorting for Single Cell and Population Genome Recovery

Abstract

1 Introduction

2 Technical Protocols and Considerations

3 Case Study: Targeted Isolation of Anaerobic Methanotrophic Archaea for Single Cell and Population Genomics

4 Summary

Acknowledgments

References

Chapter Two. Whole Cell Immunomagnetic Enrichment of Environmental Microbial Consortia Using rRNA-Targeted Magneto-FISH

Abstract

1 Introduction

2 Methods

3 Results and Discussion

4 Summary

Acknowledgments

References

Chapter Three. Coupling FACS and Genomic Methods for the Characterization of Uncultivated Symbionts

Abstract

1 Introduction

2 Fluorescence-Activated Cell Sorting

3 Metagenomic Sequencing of Uncultured Symbiont Populations

4 Determining Host Identity and Metabolism

References

Chapter Four. Optofluidic Cell Selection from Complex Microbial Communities for Single-Genome Analysis

Abstract

1 Introduction

2 Optical Tweezing for Cell Isolation

3 Optical Hardware Setup

4 Microfluidics Configuration and Setup

5 Procedure for Sorting and Amplifying Single Cells

6 Software

7 Performance and Limitations

Acknowledgments

References

Chapter Five. Quantifying and Identifying the Active and Damaged Subsets of Indigenous Microbial Communities

Abstract

1 Introduction

2 Flow Cytometry and Fluorescent Dyes

3 Experimental Procedures

4 Experimental Validation

5 Troubleshooting

Acknowledgment

References

Section II: Microbial Community Genomics—Sampling, Metagenomic Library Preparation, and Analysis

Chapter Six. Preparation of BAC Libraries from Marine Microbial Populations

Abstract

1 Introduction

2 BAC Library Construction Procedures

References

Chapter Seven. Preparation of Fosmid Libraries and Functional Metagenomic Analysis of Microbial Community DNA

Abstract

1 Introduction

2 General Considerations: Genomic Library Vector and Host

3 Construction of Fosmid Environmental Libraries

4 General Considerations for Working with Fosmids

5 Functional Screens

6 Conclusions

References

Chapter Eight. Preparation of Metagenomic Libraries from Naturally Occurring Marine Viruses

Abstract

1 On the Importance of Environmental Viruses and Viral Metagenomics

2 The DNA Viral Metagenomic Sample-to-Sequence Pipeline

3 The Library Preparation Process

4 Conclusions

Acknowledgments

References

Section III: Microbial Community Transcriptomics—Sample Preparation, cDNA Library Construction, and Analysis

Chapter Nine. Preparation and Metatranscriptomic Analyses of Host–Microbe Systems

Abstract

1 Introduction

2 Materials

3 Components

4 Experiments

5 Sequencing and Data Analysis

Acknowledgments

References

Chapter Ten. Preparation of Microbial Community cDNA for Metatranscriptomic Analysis in Marine Plankton

Abstract

1 Introduction

2 Sample Collection and RNA Preservation

3 RNA Extraction and Standardization

4 rRNA Depletion

5 RNA Amplification and cDNA Synthesis

6 Summary

Acknowledgments

References

Chapter Eleven. Sequential Isolation of Metabolites, RNA, DNA, and Proteins from the Same Unique Sample

Abstract

1 Introduction

2 Sampling and Sample Preprocessing

3 Sample Processing and Small Molecule Extraction

4 Sequential Biomacromolecular Isolation

5 Quality Control

6 Outlook

References

Chapter Twelve. Use of Internal Standards for Quantitative Metatranscriptome and Metagenome Analysis

Abstract

1 Introduction

2 Method Overview

3 DNA Template and Vector Design for Internal RNA Standards

4 mRNA Standard Preparation

5 DNA Standard Preparation

6 Internal Standard Addition

7 Internal Standard Recoveries and Quantification

8 Dataset Normalization Using Internal Standards

Acknowledgments

References

Chapter Thirteen. Sample Processing and cDNA Preparation for Microbial Metatranscriptomics in Complex Soil Communities

Abstract

1 Introduction

2 Working with RNA

3 Sampling

4 RNA Extraction of Complex Soil Communities

5 Assessing RNA Quality and Quantity

6 Soil Samples with Low Microbial Biomass

7 Removing Inhibitors

8 rRNA Subtraction/mRNA Enrichment

9 mRNA Amplification

10 cDNA Synthesis

11 Current State and Future Prospects

References

Section IV: Microbial Community Proteomics—Sampling, Sample Preparation, Spectral Analysis, and Interpretation

Chapter Fourteen. Sample Preparation and Processing for Planktonic Microbial Community Proteomics

Abstract

1 Introduction

2 Sample Collection

3 Sample Preparation

4 Extracting and Digesting Proteins

5 Mass Spectrometry

References

Chapter Fifteen. Sample Handling and Mass Spectrometry for Microbial Metaproteomic Analyses

Abstract

1 Introduction

2 Experimental Considerations and Options

3 Generalized Procedure

Acknowledgment

References

Chapter Sixteen. Molecular Tools for Investigating Microbial Community Structure and Function in Oxygen-Deficient Marine Waters

Abstract

1 Introduction

2 Exploring Oxygen Minimum Zone Community Structure

3 Detecting Oxygen Minimum Zone Proteins

Acknowledgments

References

Section V: Microbial Community Informatics: Computational Platforms, Comparative Analyses, and Statistics

Chapter Seventeen. Assembling Full-Length rRNA Genes from Short-Read Metagenomic Sequence Datasets Using EMIRGE

Abstract

1 Introduction: The Utilitarian Small Subunit rRNA Gene in Microbial Ecology

2 Overview of the EMIRGE algorithm

3 EMIRGE Outputs: Reconstructed Sequences with Estimated Abundances

4 Practical Considerations When Running EMIRGE

5 Choosing a Candidate Database of rRNA Genes

6 Conclusions and Future Outlook

References

Chapter Eighteen. Computational Methods for High-Throughput Comparative Analyses of Natural Microbial Communities

Abstract

1 Introduction

2 Sequencing Terminology

3 Sequence Processing

4 Community Analysis

5 Summary

References

Chapter Nineteen. Advancing Our Understanding of the Human Microbiome Using QIIME

Abstract

1 Introduction

2 QIIME as Integrated Pipeline of Third-Party Tools

3 PCR and Sequencing on Illumina MiSeq

4 QIIME Workflow for Conducting Microbial Community Analysis

5 Other Features

6 Recommendations

7 Conclusions

Acknowledgments

References

Chapter Twenty. Disentangling Associated Genomes

Abstract

1 Introduction

2 Sequencing

3 Data Analysis and Genome Assembly

4 Postassembly Analysis

5 Summary

Acknowledgments

References

Chapter Twenty-One. Microbial Community Analysis Using MEGAN

Abstract

1 Introduction

2 Taxonomic Analysis

3 Functional Analysis

4 Sequence Alignment

5 Comparison of Samples

6 Conclusion

References

Chapter Twenty-Two. A Metagenomics Portal for a Democratized Sequencing World

Abstract

1 Introduction

2 Pipeline and Technology Platform

3 Web Interface

4 How to Drill Down Using the Workbench

5 MG-RAST Downloads

6 Discussion

7 Future Work

Acknowledgments

References

Chapter Twenty-Three. A User’s Guide to Quantitative and Comparative Analysis of Metagenomic Datasets

Abstract

1 Introduction

2 How to Assemble a Metagenomic Dataset

3 How to Determine the Fraction of the Community Captured in a Metagenome

4 How to Identify the Taxonomic Identity of a Metagenomic Sequence

5 How to Determine Differentially Abundant Genes, Pathways, and Species

6 Limitations and Perspectives for the Future

Acknowledgments

References

Author Index

Subject Index