High-density sequencing applications in microbial molecular genetics / edited by Agamemnon J. Carpousis.

Format:

Book

Contributor:

Carpousis, Agamemnon J., editor.

Series:

Methods in enzymology ; Volume 612.

Methods in enzymology, 0076-6879 ; Volume 612

Language:

English

Subjects (All):

Molecular genetics.

Physical Description:

1 online resource (576 pages).

Place of Publication:

Cambridge, MA : Academic Press, an imprint of Elsevier, [2018]

Summary:

High-Density Sequencing Applications in Microbial Molecular Genetics, Volume 612 in the Methods of Enzymology series provides the latest on the high-density sequencing of DNA and cDNA libraries and how they have revolutionized contemporary research in biology. Methods permitting tens of millions of sequence reads in a single experiment have paved the way to genome-wide studies that are contributing to our understanding of the complexity of living systems. Chapters in this updated volume include Characterizing the role of exoribonucleases in the control of microbial gene expression: Differential RNA seq., Conformational studies of bacterial chromosomes by high-throughput sequencing methods, Measuring mRNA degradation, and more.Addition sections cover Global recognition patterns of bacterial RNA-binding proteins, High-resolution profiling of NMD targets, and the Generation of a metagenomic 3C/Hi-C library of human gut microbiota, Genome-wide mapping of yeast retrotransposons integration target sites, Measuring protein synthesis rates, Finding unsuspected partners of small RNAs with new screening approaches, Use of multiplexed transcriptomics to define the relationship between promoter sequence and transcription output, RNA-based control of quorum sensing in Vibrio cholerae, amongst other highly regarded topics.- Detail methods used in research articles that were recently published in leading journals- Provides the latest on the high-density sequencing of DNA and cDNA libraries and how they have revolutionized contemporary research in biology

Contents:

Front Cover

High-Density Sequencing Applications in Microbial Molecular Genetics

Copyright

Contents

Contributors

Preface

Chapter One: Characterizing the Role of Exoribonucleases in the Control of Microbial Gene Expression: Differential RNA-Seq

1. Overview of Exoribonucleases

2. Overview of Differential RNA-Seq

3. RNA Extraction

4. Bioinformatics Analysis

4.1. Mapping of Reads

4.2. Transcript Expression Quantification

4.3. Differential Expression Analysis

4.4. Functional Annotation Analysis

5. Experimental Validation

5.1. qPCR

5.2. Motility Assays

6. Conclusion

Acknowledgments

References

Chapter Two: Conformational Studies of Bacterial Chromosomes by High-Throughput Sequencing Methods

1. Introduction

1.1. Chromosome Conformation

1.2. DNA-Protein Interactions

1.3. Combining NGS to 3C and ChIP Approaches

2. 3C-Seq: The Protocol

2.1. Buffers and Reagents

2.2. Cross-Linking of Cell Cultures

2.3. 3C Library Preparation

2.3.1. Cell Lysis

2.3.2. RE Digestion

2.3.3. Ligation

2.3.4. DNA Purification

2.4. Notes

3. ChIP-Seq: The Protocol

3.1. Buffers and Reagents

3.2. Cross-Linking of Cell Cultures

3.3. Cell Lysis

3.4. Batch Immunoprecipitation of FLAG Fusions Proteins

3.5. Elution of the Immunoprecipitated Protein

3.6. Purification of the Coimmunoprecipitated DNA

3.7. Notes

4. Data Processing and Analysis

4.1. Analysis of 3C-Seq Data

4.2. Analysis of ChIP-Seq Data

5. Conclusion

Chapter Three: Large-Scale Measurement of mRNA Degradation in Escherichia coli: To Delay or Not to Delay

2. Measuring mRNA Degradation Rates

2.1. Methods

2.2. Rifampicin

2.3. Practical Considerations

3. Genome-Wide mRNA Quantification

3.1. mRNA Extraction and Quantification.

3.2. Normalization

4. mRNA Half-Life Estimation

4.1. Decay Models

4.2. Accuracy Criteria of the Estimation

5. Effect of the Delay Before the Onset of Exponential Decay on mRNA Half-Life Estimation

5.1. Datasets of mRNA Levels Over the Time

5.2. Estimations of the Delay Before the Onset of the mRNA Exponential Decay

5.3. Comparison of mRNA Half-Lives Estimated With and Without a Delay

5.4. Correlation Between mRNA Half-Lives Estimated With and Without a Delay

5.5. Influence of the Delay Extent

5.6. Validation on RT-qPCR mRNA Quantification Datasets

6. Concluding Remarks

Chapter Four: FASTBAC-Seq: Functional AnalysiS of Toxin-Antitoxin Systems in BACteria by Deep Sequencing

2. Materials

2.1. Molecular Biology Equipment

2.2. General Equipment for Bacterial Cultures

2.3. E. coli-Specific Equipment

2.4. H. pylori-Specific Equipment

2.5. Molecular Biology Reagents

2.6. E. coli Strains and Cell Culture

2.7. H. pylori Strains and Cell Culture

3. Methods: A High-Throughput Genetic Selection of Intrinsic Toxicity Suppressors in Six Steps

3.1. Step 1: Deletion of the TA Locus Using the rpslCj-erm Cassette

3.1.1. Creation of a rpslCj-erm-Based TA-Deletion Cassette by PCR Assembly

3.1.1.1. Notes

3.1.2. H. pylori Natural Transformation and Isolation of Recombinant Strains

3.2. Step 2: Creation of TA and Toxin (T) Locus by PCR Assembly

3.2.1. TA Locus Subcloning in E. coli

3.2.1.1. Notes

3.2.2. Inactivation of the Antitoxin Promoter by SDM PCR

3.2.2.1. Notes

3.2.3. Reconstruction of the TA Locus by PCR Assembly

3.2.3.1. Notes

3.3. Step 3: Natural Transformation of H. pylori With the PCR-Generated TA Loci

3.3.1. Notes

3.4. Step 4: Selection of Transformants

3.4.1. Determining the Transformation Efficiency.

3.4.1.1. Notes

3.4.2. Differentiating Transformants From Revertants: The StrR/ErmR Screen

3.5. Step 5: Sample Preparation and Sequencing by Illumina Paired-End Approach

3.5.1. Pooling of Isolated Strains and Genomic DNA Extraction

3.5.2. The Nested PCR Strategy

3.5.3. Illumina Paired-End Sequencing

3.5.3.1. Notes

3.6. Step 6: Computational Analysis of the NGS Data

3.6.1. Read Reprocessing and Assembly

3.6.1.1. Notes

3.6.2. Read Alignment

3.6.2.1. Notes

3.6.3. Statistical Analysis

3.6.4. Nucleotide-Specific Analysis

3.6.5. Positional Analysis

3.6.6. Differential Expression Analysis

3.6.6.1. Notes

3.6.7. Generate Plots of Analysis Results

3.6.7.1. Notes

4. A Case Study: Decoding AapA3 Peptide Toxicity Determinants With Nucleotide Resolution

4.1. Comprehensive Understanding of Suppressor Mutations

4.2. Domain Identification in the Small Toxic Protein AapA3

5. Conclusions

5.1. Important Requirements

5.1.1. The Importance of Sequencing "Wild-Type" Transformed Strains

5.1.2. The Importance of Analyzing (At Least) Three Independent Biological Replicates

5.2. Advantages

5.2.1. Fast, Systematic, High-Throughput, and Cheap

5.2.2. Study of the Endogenous Expression of TA Systems

5.3. Limitations

5.3.1. Exclusive Identification of Loss-of-Function Mutations

5.3.2. Only Single-Nucleotide Mutations

5.4. Perspectives

5.4.1. Adapting FASTBAC-Seq to Other Bacterial Species

5.4.2. Applying FASTBAC-Seq to Study Other TA Systems

Chapter Five: The Challenges of Genome-Wide Studies in a Unicellular Eukaryote With Two Nuclear Genomes

2. Protocol: Separating the Micronucleus From the Macronucleus

2.1. MIC Purification

2.1.1. Buffers and Solutions

2.1.2. Cell Culture

2.1.3. Cell Centrifugation.

2.1.4. Cell Lysis

2.1.5. Centrifugation Step 1

2.1.6. Centrifugation Step 2

2.1.7. Freezing

2.2. Sorting by Flow Cytometry

2.3. Quality Control: Flow Imaging and High-Throughput Sequencing

2.3.1. Flow Cell Imaging

2.3.2. Genomic DNA Extraction and Sequencing

3. Analysis of Sequence Data

3.1. Preprocessing of Sequencing Reads

3.2. Quality Control of Sequencing Reads Using k-mer Analysis (QC1)

3.3. Removing MAC Reads That Could Compromise Assembly (MAC Filter)

3.4. Assembly Strategies

3.5. Quality Control of the Assembly (QC2)

4. Perspectives and Limits

Chapter Six: CLIP-Seq in Bacteria: Global Recognition Patterns of Bacterial RNA-Binding Proteins

1.1. Studying RNA-Protein Interactions

2. Method Overview

2.1. CLIP: History and Development

2.2. CLIP: Strengths and Limitations

2.3. CLIP in Bacteria

3. Experimental Method

3.1. General Considerations

3.2. Equipment

3.3. Materials

3.4. Solutions and Buffers

3.5. Procedure

3.5.1. Growth of Bacteria, UV Cross-linking, and Cell Lysis

3.5.2. Immunoprecipitation

3.5.3. RNase Treatment, Dephosphorylation, and Radioactive Labeling

3.5.4. SDS-PAGE and Transfer to Membrane

3.5.5. RBP Digestion and RNA Extraction

3.5.6. Adaptor Ligation and cDNA Synthesis

3.5.7. Initial cDNA Library Amplification to Optimize PCR Cycle Number

3.5.8. cDNA Library Amplification With Optimal PCR Cycle Number

3.5.9. Optional: Reamplification of DNA Libraries

3.5.10. Sequencing

3.6. Notes

Chapter Seven: High-Resolution Profiling of NMD Targets in Yeast

2. Construction of Strand-Specific RNA-Seq Libraries From Wild-Type and NMD-Deficient Yeast Cells

2.1. Yeast Strains and Growth Conditions

2.1.1. Equipment.

2.1.2. Buffers and Reagents

2.1.3. Procedure

2.1.4. Notes

2.2. Total RNA Isolation From Yeast Cells

2.2.1. Equipment

2.2.2. Buffers and Reagents

2.2.3. Procedure

2.2.4. Notes

2.3. Removal of Genomic DNA Contamination by DNase Treatment

2.3.1. Equipment

2.3.2. Buffers and Reagents

2.3.3. Procedure

2.3.4. Notes

2.4. Removal of rRNAs From the Total RNA Samples by Ribo-Zero Treatment

2.4.1. Equipment

2.4.2. Buffers and Reagents

2.4.3. Procedure

2.4.3.1. Prepare the Magnetic Beads

2.4.3.2. Hybridize the rRNA Removal Probes to the rRNAs in the Total RNA Sample

2.4.3.3. Remove rRNAs From the Total RNA Sample

2.4.3.4. Purify the rRNA-Depleted Total RNA Sample by Ethanol Precipitation

2.4.3.5. Determine Yield and Quality of the rRNA-Depleted Total RNA Samples

2.4.4. Notes

2.5. Construction of RNA-Seq Libraries

2.5.1. Equipment

2.5.2. Buffers and Reagents

2.5.3. Procedure

2.5.3.1. RNA Fragmentation

2.5.3.2. First-Strand cDNA Synthesis

2.5.3.3. Second-Strand cDNA Synthesis

2.5.3.4. Purification of Double-Stranded cDNA Fragments

2.5.3.5. 3′ Adenine Addition to the cDNA Fragments

2.5.3.6. Ligation of Indexing Adapters

2.5.3.7. Removal of Free Adapters From the Ligation Products

2.5.3.8. PCR Amplification of Library cDNA Fragments

2.5.3.9. Purification of PCR-amplified Library DNA Fragments

2.5.3.10. Analyzing the Quality and the Yield of RNA-Seq Libraries

2.5.4. Notes

3. Sequencing of Total RNA cDNA Libraries From Wild-Type and NMD-Deficient Yeast Cells

4. Identification of Transcripts Targeted by NMD

4.1. Software Packages

4.2. Procedure

4.2.1. Constructing the Reference Transcriptome

4.2.2. Assessing the Quality and the General Mapping Statistics of RNA-Seq Libraries.

4.2.3. Mapping Sequence Reads to the Reference Transcriptome and Estimating the Reads Mapped to Individual Transcripts.

Notes:

Description based on print version record.

ISBN:

0-12-815994-4

0-12-815993-6