Microbial Metagenomics, Metatranscriptomics, and Metaproteomics - 1st Edition - ISBN: 9780124078635, 9780124079274

Microbial Metagenomics, Metatranscriptomics, and Metaproteomics, Volume 531

1st Edition

Serial Volume Editors: Ed DeLong
eBook ISBN: 9780124079274
Hardcover ISBN: 9780124078635
Imprint: Academic Press
Published Date: 1st October 2013
Page Count: 688
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Table of Contents

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


Description

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers microbial metagenomics, metatranscriptomics, and metaproteomics, and includes chapters on such topics as in-solution FISH for single cell genome preparation, preparation of BAC libraries from marine microbial community DNA, and preparation of microbial community cDNA for metatranscriptomic analysis in marine plankton.

Key Features

  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Covers microbial metagenomics, metatranscriptomics, and metaproteomics
  • Contains chapters on such topics as in-solution fluorescence in situ hybridization (FISH) for single cell genome preparation, preparation of BAC libraries from marine microbial community DNA, and preparation of microbial community cDNA for metatranscriptomic analysis in marine plankton

Readership

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.


Details

No. of pages:
688
Language:
English
Copyright:
© Academic Press 2013
Published:
Imprint:
Academic Press
eBook ISBN:
9780124079274
Hardcover ISBN:
9780124078635

About the Serial Volume Editors

Ed DeLong Serial Volume Editor