Microbial Environmental Genomics (MEG)

Preface
Contents
Contributors
Chapter 1: Metabarcoding Approaches for Soil Eukaryotes, Protists, and Microfauna
1 Introduction
2 Materials
2.1 DNA Extraction
2.1.1 Soil DNA Extraction for the Entire Eukaryome
2.1.2 Nematode Isolation From Soils with Subsequent DNA Extraction
2.1.3 Soil DNA Extraction for Microarthropods
2.2 Amplicon Preparation for Metabarcoding
2.2.1 All Eukaryotes with Special Focus on Protists and Nematodes
2.2.2 Microarthropods
2.3 Software for Bioinformatic Processing
2.3.1 In a Bash Operating System (Linux or macOS)
2.3.2 In R v3.4 and Beyond (https://cran.r-project.org/)
2.3.3 Example Data and Scripts
3 Methods
3.1 Soil DNA Extraction
3.1.1 Soil Total DNA Extraction for the Entire Eukaryome
3.1.2 DNA Extraction of Nematodes
3.1.3 Soil Extracellular DNA Extraction for Microarthropods
3.2 Amplicon Preparation
3.2.1 Metabarcoding of the Soil Eukaryome, Including Protists and Nematodes
3.2.2 Metabarcoding of Microarthropods
3.3 Bioinformatic Analyses
3.3.1 Metabarcoding of All Eukaryotes with a Focus on Protists
3.3.2 Metabarcoding of Nematodes
3.3.3 Metabarcoding of Microarthropods
4 Notes
References
Chapter 2: Metabarcoding of the Three Domains of Life in Aquatic Saline Ecosystems
1 Introduction
2 Materials
3 Methods
3.1 Sampling
3.2 Sample Processing and Preservation of Water Samples
3.3 Nucleic Acid Extraction and Purification
3.4 PCR Amplification and Sequencing of the Taxonomic Gene Markers
3.4.1 Amplicon Libraries Construction by Two-Step PCR
3.4.2 Amplicon Library Sequencing
3.5 Bioinformatic Analyses
4 Notes
References
Chapter 3: Sample Preparation for Fungal Community Analysis by High-Throughput Sequencing of Barcode Amplicons
1 Introduction
2 Materials
2.1 DNA Preparation Components
2.2 Amplicon Preparation Components
3 Methods
3.1 Sample Preparation
3.2 DNA Extraction and Purification
3.2.1 DNA Extraction Using the CTAB Method
3.2.2 Optional: Purification with the Wizard DNA Clean-Up System (Promega)
3.2.3 DNA Quantification and Purity Check by NanoDrop
3.3 PCR Optimization
3.3.1 Test of Template Concentrations and Cycle Numbers
3.3.2 PCR Products Visualized by Gel Electrophoresis
3.4 Production of Tagged ITS Amplicons
3.4.1 PCRs with Sample-Tagged ITS Primers
3.4.2 Clean PCR Products with the AMPure Kit
3.5 Amplicon Mixing and Sequencing
3.5.1 Quantification of Double-Stranded DNA with the Qubit dsDNA HS Assay Kit
3.5.2 Calculating the Concentration of dsDNA in Each Sample
3.5.3 Make an Equal Mix of All Amplicons
3.5.4 Clean Amplicon Mix with Cycle-Pure Kit (Omega)
3.5.5 PCR Product Quantification and Quality Control by Bioanalyzer (Agilent Tech)
4 Notes
References
Chapter 4: Characterization of Seed Mycobiota Using Culture-Dependent and Culture-Independent Approaches
1 Introduction
2 Materials
2.1 Culture-Based Characterization
2.1.1 Isolation from Seed Material
2.1.2 Archiving Fungal Isolates for Long-Term Use
2.1.3 DNA Extraction
2.1.4 Amplicon Preparation for Sanger Sequencing
2.2 Culture-Free Characterization
2.2.1 DNA Extraction and Purification
2.2.2 Amplicon Preparation for Metabarcoding
3 Methods
3.1 Culture-Based Characterization
3.1.1 Isolation from Seed Tissue
3.1.2 Archiving Isolates for Long-Term Use
3.1.3 DNA Extraction
3.1.4 Amplicon Preparation for Sanger Sequencing
3.2 Culture-Free Characterization
3.2.1 DNA Extraction
3.2.2 Amplicon Preparation for Illumina MiSeq
4 Notes
References
Chapter 5: Tissue Cultivation, Preparation, and Extraction of High Molecular Weight DNA for Single-Molecule Genome Sequencing ...
1 Introduction
2 Materials
2.1 Collection and Preparation of Fungal Fruiting Bodies for Extraction
2.2 Growth and Harvesting of Fungal Mycelium on Solid Media with Cellophane Overlay
2.3 Growth and Harvesting of Fungal Mycelium in Liquid Media
2.4 Genomic DNA Extraction
2.5 Quality Assessment of Genomic DNA
2.5.1 Assessing Genomic DNA Quantity by Qubit Fluorometer
2.5.2 Assessing the Purity of Genomic DNA
2.5.3 Assessing the Presence of Inhibitors in Genomic DNA
2.5.4 Sizing of High Molecular Weight DNA and Evaluating Degradation
2.6 DNA Clean-Up Using Qiagen Genomic Tips 500/G
3 Methods
3.1 Collection and Preparation of Fungal Fruiting Bodies for Extraction
3.2 Growth and Harvesting of Fungal Mycelium on Solid Media with Cellophane Overlay
3.3 Growth and Harvesting of Fungal Mycelium in Liquid Media
3.4 Genomic DNA Extraction (Protocol Modified from Fulton et al., See Note 17)
3.5 Quality Assessment of Genomic DNA
3.5.1 Assessing Genomic DNA Quantity by Qubit Fluorometer
3.5.2 Assessing the Purity of Genomic DNA
3.5.3 Assessing the Presence of Inhibitors in Genomic DNA
3.5.4 Sizing of High Molecular Weight DNA and Evaluating Degradation
3.6 DNA Clean-up Using Qiagen Genomic Tips 500/G
4 Notes
References
Chapter 6: Analysis of Ancient Microbial DNA
1 Introduction
2 Materials
2.1 DNA Extraction Reagents
2.2 qPCR Reagents
2.3 Library Preparation for PCR Products
2.4 Double-Stranded DNA Library Preparation
2.5 Single-Stranded DNA Library Preparation
2.6 Equipment (Other Than Common Devices for Molecular Biology Laboratories)
3 Methods (See Note 12)
3.1 DNA Extraction and Purification
3.2 DNA Amplification
3.3 Library Preparation for PCR Products
3.4 Double-Stranded DNA Library Preparation
3.5 Single-Stranded DNA Library Preparation
4 Notes
References
Chapter 7: Whole-Genome Sequencing of Bacterial Endophytes From Fresh and Preserved Plant Specimens
1 Introduction
2 Materials
2.1 Sampling
2.2 DNA Extraction
2.2.1 DNA Extraction of Fresh or Silica-Dried Plant Samples
2.2.2 DNA Extraction of Preserved Plant Samples
2.3 Short DNA Sequencing Library Building
2.4 Bioinformatics Analysis of Leaf Metagenomes
3 Methods
3.1 Sampling
3.2 DNA Extraction From Fresh or Silica-Dried Leaves
3.3 DNA Extraction From Preserved Plant Tissue (Herbarium Specimens)
3.4 Preparing Sequencing Libraries From Degraded DNA
3.4.1 Preparing Adapter Mix
3.4.2 DNA End Repair
3.4.3 DNA Adapter Ligation
3.4.4 PCR Fill-In
3.4.5 Library Amplification and Indexing
3.5 Bioinformatics Analysis
3.5.1 Preprocessing of Sequencing Reads
3.5.2 Taxonomic Assignment of Reads
3.5.3 Metagenome Assembly
3.5.4 Metagenome Binning
3.5.5 Annotation of Bins
4 Notes
References
Chapter 8: Investigating the Bacterial and Fungal Communities Involved in Dead Biomass Degradation in Forest Soils
1 Introduction
2 Materials
2.1 Stable Isotope Probing
2.2 Gene-Targeted Metagenomics and Metatranscriptomics
3 Methods
3.1 SIP to Identify Cellulose-Utilizing Fungi
3.2 Gene-Targeted Metagenomics and Metatranscriptomics to Identify Exocellulase Producers
4 Notes
References
Chapter 9: Stable Isotope Probing-RNA Strategy to Study Plant/Fungus Interactions
1 Introduction
2 Material
2.1 Material Common to Methods 1 and 2
2.2 Specific Material of Method 1
2.3 Specific Material of Method 2
3 Method
3.1 Method 1. Identification of Fungi Interacting with Their Host Plant by SIP-RNA
3.2 Method 2. Analysis of the Carbon Transfer Intensity From the Plant to Fungi by SIP-RNA
4 Notes and Troubleshooting
5 Conclusions and Prospects
References
Chapter 10: Targeted 16S rRNA Gene Capture by Hybridization and Bioinformatic Analysis
1 Introduction
2 Materials
2.1 Reagents and Kits
2.2 Buffers and Solutions
2.3 Oligonucleotides (Probes and Primers)
2.4 Equipment
2.5 Bioinformatic Tools
3 Methods
3.1 Synthesis of Biotinylated RNA Capture Probes
3.2 Library Preparation (550 bp Insert)
3.3 Library Amplification
3.4 Library Purification
3.5 Gene Capture by Hybridization
3.6 Washing and Elution of Captured Library
3.7 Purification and Amplification of Captured Library
3.8 Second Cycle of Gene Capture by Hybridization
3.9 Bioinformatic Analysis
4 Notes
References
Chapter 11: Construction of a Transposon Mutant Library in the Pathogen Agrobacterium tumefaciens C58 and Identification of Ge...
1 Introduction
2 Materials
2.1 Transposon Library Construction
2.1.1 Bacterial Culture Conditions
2.1.2 Bacterial Conjugation
2.2 Growth of the Transposon Mutant Library in Rich Media (T0 Population)
2.3 Growth of the Transposon Mutant Library on Minimal Medium
2.4 Growth of the Transposon Mutant Library in Tomato Plant Tumors
2.4.1 Plant Growth
2.4.2 Inoculation of Tomato Plants
2.4.3 Collection of Tomato Plant Tumors
2.5 Transposon Library DNA Preparation
2.6 Transposon Library DNA Sequencing and Related Sequence Analyses
2.6.1 Transposon Library DNA Sequencing
2.6.2 Sequence Analyses Using ARTIST
2.7 Building-up a Unique List of Essential Genes
2.8 Visual Exploration of Fitness Data
3 Methods
3.1 Transposon Library Construction
3.1.1 Bacterial Culture Conditions
3.1.2 Bacterial Conjugation
3.2 Growth of the Transposon Mutant Library in Rich Media (T0 Population)
3.3 Growth of the Transposon Mutant Library on Minimal Media
3.4 Growth of the Transposon Mutant Library in Tomato Plant Tumors
3.4.1 Plant Growth
3.4.2 Inoculation of Tomato Plants
3.4.3 Collection of Tomato Plant Tumors
3.5 Transposon Library DNA Preparation
3.6 Transposon Library DNA Sequencing and Sequence Analysis
3.6.1 Transposon Library DNA Sequencing
3.6.2 Sequence Analysis Using ARTIST
EL-ARTIST Analysis
CON-ARTIST Analysis
3.7 Building-up a Unique List of Essential Genes
3.8 Visual Exploration of Fitness Data
4 Notes
References
Chapter 12: High-Throughput Screening of Fosmid Libraries for Increased Identification of Novel N-Acyl Homoserine Lactone Degr...
1 Introduction
2 Materials
2.1 AHL and Culture Medium for the Enrichment Method
2.2 DNA Extraction, Purification, and Manipulation
2.3 Fosmid Library Construction
2.4 Fosmid Library Screening
2.5 Equipment
3 Method
3.1 Sample Recovery and Preparation
3.1.1 Direct (Method #1)
3.1.2 Enrichment of AHL Degrading Bacteria (Method #2)
3.2 DNA Extraction
3.2.1 Total DNA Extraction From Soil (Method #1)
3.2.2 Total DNA Extraction of Enrichment Cultures (Method #2)
3.3 Purification of High-Molecular Weight DNA
3.4 Fosmid Library Construction
3.5 Functional Screenings
3.5.1 AHL Production Screening
3.5.2 AHL Degradation Screening
3.5.3 AHL Detection
4 Notes
References
Chapter 13: Analyzing Prokaryotic Transcriptomics in the Light of Genome Data with the MicroScope Platform
1 Introduction
2 Prerequisite for Transcriptomics Analysis
2.1 User Prerequisites
2.2 MicroScope Genome Annotation Process
2.2.1 Syntactic Annotation
2.2.2 Functional Annotation
2.2.3 Relational Annotation
3 Methods
3.1 Data File Validation
3.2 Raw Read Quality Control
3.3 Strandedness Checking
3.4 Mapping with Virtual Trimming
3.5 Contamination Screening
3.6 Raw Reads Counts
3.7 rRNA Read Quantification
3.8 Normalization
3.9 Sample Quality Control
3.10 Differential Analysis
4 Web Interface
4.1 Delivery of Service Pages
4.2 Project Page
4.3 Mapping Overview
4.4 Raw Read Count
4.5 Differential Analysis Page
4.5.1 Setting Up the Displayed Results
4.5.2 Differential Analysis Table
4.5.3 Analysis Summary and Plots
4.6 Tools for Functional Analysis
4.6.1 Using the Keyword Search with Gene Carts
4.6.2 EGGNOG Classification
4.6.3 MicroCyc Pathway Results
4.6.4 Integrated Genome Viewer
5 Notes
References
Chapter 14: MycoCosm, the JGI´s Fungal Genome Portal for Comparative Genomic and Multiomics Data Analyses
1 Introduction
2 Finding Genomes
2.1 Search
2.2 Navigator
2.3 Phylo-/EcoGroups
2.4 Interactive Tree
3 Individual Portal Tools
3.1 Search
3.2 BLAST
3.3 Browser
3.3.1 Multi-Omics
3.4 Protein Page
3.5 Synteny
3.6 MCL Clustering
3.7 Mitochondrion
3.8 Download
4 Comparative Annotations
4.1 GO
4.2 Pfam
4.3 KEGG
4.4 KOG
4.5 Secondary Metabolism
4.6 CAZymes
4.7 Peptidases
4.8 Transporters
4.9 Transcription Factors
5 Additional Resources
References
Chapter 15: Investigating Endobacteria that Thrive Within Mucoromycota
1 Introduction
2 Materials
2.1 Isolation of Mortierellaceae Fungi
2.1.1 Isolation of Mortierellaceae Using the Soil Plate Method
2.1.2 Isolation of Mortierellaceae from Plant Roots
2.1.3 Isolation of Mortierellaceae Using the Surface Washing Method
2.1.4 Isolation of Mortierellaceae Using the Moist Chamber Method
2.2 Decontamination of Bacteria from the Hyphal Surface
2.3 Efficient Removal of Endobacteria from the Fungal Host
2.3.1 Clearing Endobacteria from Germinated Sporangiospores of the Fungal Host
2.3.2 Clearing Endobacteria from the Fungal Host Mycelium of the Fungal Host
2.4 Maintaining Fungal Lines
2.5 Sporulation of Mortierellaceae
2.6 Microscopy
2.6.1 Fluorescent In-Situ Hybridization (FISH)
2.6.2 Transmission Electron Microscopy (TEM)
2.7 Molecular Analyses
2.7.1 DNA Extraction
2.7.2 PCR
2.7.3 Cloning
2.7.4 Real-Time qPCR
2.8 Isolation of Mycoavidus cysteinexigens from Its Fungal Host
3 Methods
3.1 Isolation of Mortierellaceae
3.1.1 Isolation of Mortierellaceae from Soil with a Modified Version of the Soil-Plate Method
3.1.2 Isolation of Mortierellaceae from Plant Roots
3.1.3 Isolation of Mortierellaceae from Plant Materials by Surface Washing Method
3.1.4 Isolation of Fungi from Soil by Using the Moist Chamber Method
3.2 Decontamination of Bacteria from the Hyphal Surface
3.3 Obtaining an Isogenic ``Cured´´ Line: Efficient Removal of Endobacteria from the Fungal Host
3.3.1 Clearing Endobacteria from Germinated Sporangiospores of the Fungal Host
3.3.2 Clearing Endobacteria from the Mycelium of the Fungal Host
3.4 Maintaining Fungal Lines
3.5 Sporulation of Mortierellaceae
3.6 Microscopy
3.6.1 Fluorescent In-Situ Hybridization (FISH)
3.6.2 Transmission Electron Microscopy (TEM)
3.7 Molecular Analyses
3.7.1 DNA Extraction
3.7.2 Molecular Detection of BRE and MRE
3.7.3 Real-Time qPCR
3.8 Isolation of Mycoavidus cysteinexigens from Its Fungal Host
4 Notes
References
Chapter 16: Confocal Laser Scanning Microscopy Approach to Investigate Plant-Fungal Interactions
1 Introduction
2 Materials
2.1 General Laboratory Facilities
2.2 Chemicals
2.3 Sample Collection
2.4 Sample Staining
2.5 Microscopy Observation
3 Methods
3.1 Preparation of Root Samples
3.2 Root Staining
3.2.1 Full-Mount Microscopy
3.2.2 Microscopy of Cross Sections
3.3 CLSM Observations (Fig. 2)
3.4 Fungal Quantification
3.4.1 Quantification of Fungal Colonization
3.4.2 Assessment of Hartig Net Formation
4 Notes
References
Chapter 17: Visualization of Fungi During Wood Colonization and Decomposition by Microscopy: From Light to Electron Microscopy
1 Introduction
2 Materials
3 Methods
3.1 Production of Material
3.1.1 In Situ Assays
3.1.2 In Vitro Assay
3.2 Sample Preparation Before Imaging
3.3 Dark Field Epimicroscopy Imaging
3.4 Wide Field Transmitted Light Microscope Imaging
3.5 Epifluorescence and Laser Scanning Confocal Imaging
3.5.1 Labelling of Fungi Prior Imaging
3.5.2 Epifluorescence Imaging
3.5.3 Confocal Imaging
Regular Imaging
Spectral Deconvolution
3.6 Scanning Electron Microscopy Imaging
3.6.1 Coating (see Note 15)
Coating for Regular SEM Imaging
Coating for High-Resolution Imaging with Field Emission Gun
3.6.2 Regular SEM Imaging and High-Resolution Imaging with Field Emission Gun (FEG) Scanning Electron Microscope
3.7 Correlative Imaging
3.7.1 Sample Preparation
3.7.2 Imaging (see Note 21)
4 Notes
References
Index