Marine Genomics: Methods and Protocols (Methods in Molecular Biology, 2498)

Dedication
Preface
Contents
Contributors
Chapter 1: Mitochondrial Genome of Nonmodel Marine Metazoans by Next-Generation Sequencing (NGS)
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 DNA Fragmentation
			2.2.1 Mechanical Fragmentation
			2.2.2 Enzymatic Fragmentation
			2.2.3 Fragmented DNA Purification
		2.3 Library Preparation
		2.4 DNA Extraction, DNA Fragmentation, and Library Validation
			2.4.1 Size/Quality Validation Using an Automated Capillary Electrophoresis System
			2.4.2 Size/Quality Validation Using Agarose Gel Electrophoresis
			2.4.3 Double-Stranded DNA (dsDNA) Quantification
		2.5 Pool Validation
			2.5.1 Pool Size/Quality Validation
			2.5.2 Pool Concentration Quantification
		2.6 Bioinformatic Analyses
	3 Methods
		3.1 DNA Extraction
		3.2 DNA Extraction Validation
			3.2.1 DNA Size/Quality Control Using an Automated Capillary Electrophoresis System
			3.2.2 DNA Size/Quality Control Using Agarose Gel Electrophoresis
			3.2.3 dsDNA Extract Quantification
		3.3 DNA Fragmentation
			3.3.1 DNA Mechanical Fragmentation
			3.3.2 DNA Enzymatic Fragmentation
			3.3.3 Fragmented DNA Purification
		3.4 Fragmented DNA Validation
		3.5 Library Preparation
		3.6 Pool Validation
			3.6.1 qPCR
		3.7 High-Throughput Sequencing
		3.8 Bioinformatic Analyses
	4 Notes
	References
Chapter 2: Genome-Wide DNA Methylation Protocol for Epigenetics Studies
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
	3 Methods
		3.1 Digestion of Genomic DNA
		3.2 End-Repair (ER) Reaction
		3.3 A-tailing (A-t) Reaction
		3.4 Adapter Annealing
		3.5 Adapter Ligation (AL) Reaction
		3.6 Analytical PCRI
		3.7 Size Selection
		3.8 Analytical PCRII
		3.9 Bisulfite Conversion
		3.10 Enrichment PCR
		3.11 Prepare Libraries for Sequencing and for Data Analysis
	4 Notes
	References
Chapter 3: Transcriptome Mining to Identify Genes of Interest: From Local Databases to Phylogenetic Inference
	1 Introduction
	2 Bioinformatic Pipeline
		2.1 Creating a Protein Database
		2.2 Sequence Alignment
		2.3 Trimming of Spurious Regions
		2.4 Evolution Models Selection and Phylogenetic Inference
	3 Notes
	References
Chapter 4: Detecting Structural Variants and Associated Gene Presence-Absence Variation Phenomena in the Genomes of Marine Org...
	1 Introduction
	2 Materials
		2.1 Target Selection
		2.2 Software
	3 Methods
		3.1 Allelic Structural Variation Detection Within Assembled Genomes
		3.2 Gene Presence-Absence Variation Detection and Analysis
		3.3 PAV Gene Functional Enrichment Analysis
	4 Notes
	References
Chapter 5: From Sequences to Enzymes: Comparative Genomics to Study Evolutionarily Conserved Protein Functions in Marine Micro...
	1 Introduction
	2 Materials
	3 Methods
		3.1 Retrieving Closely Related Amino Acid Sequences from the Database
		3.2 Retrieving 16S rRNA Gene Sequences for the Selected Organisms from the Database
		3.3 Amino Acid Sequence Alignment and Evaluation of Conserved Regions
		3.4 Nucleotide Sequences Alignment
		3.5 Phylogenetic Trees Construction
		3.6 Phylogenetic Trees Reconciliation (Optional Step)
		3.7 Identification of the Genomic Context for the Protein-Coding Gene of Interest
		3.8 Protein 3D Structure Homology Modeling
	4 Notes
	References
Chapter 6: VenomFlow: An Automated Bioinformatic Pipeline for Identification of Disulfide-Rich Peptides from Venom Arsenals
	1 Introduction
	2 Implementation
	3 Notes
	4 Discussion
	References
Chapter 7: Population Genomics Analysis with RAD, Reprised: Stacks 2
	1 Introduction
		1.1 The RAD Family of Protocols
		1.2 Top Molecular Impediments to Good Results
		1.3 Stacks Version 2: De Novo and Reference-Based RAD Analysis
		1.4 Stacks Haplotypes Versus SNPs
	2 Methods
		2.1 Presequencing: RADseq Experimental Design
		2.2 Data Set Used in this Protocol
		2.3 Simulations
			2.3.1 Calculating an Estimated Tally of RAD Loci
			2.3.2 Estimating Sequencing Coverage
		2.4 Cleaning and Demultiplexing Data
			2.4.1 Assaying the Processing and Cleaning of the Raw Data
		2.5 De Novo Analysis
			2.5.1 Parameter Optimization
			2.5.2 Core De Novo Pipeline Execution
			2.5.3 Assaying the Effectiveness of De Novo Assembly
		2.6 Reference-Aligned Analysis
			2.6.1 Assaying the Effectiveness of a Reference-Based Assembly
		2.7 Integrated Analysis
		2.8 Populations
			2.8.1 Assessing the Populations Results
			2.8.2 Post-analysis Directions After Stacks
				SNP-Based Population Structure
				Haplotype-Based Population Structure
				Divergence Statistics
				Linkage Disequilibrium
		2.9 Triaging Negative Results
			2.9.1 Should I Trim Data?
			2.9.2 Controlling the Stringency for Calling Genotypes at Low Coverage
			2.9.3 Removing Bad Apples
	Appendix
	Plot Library Demultiplexing and Processing
	De Novo Parameter Optimization Shell Loop
	Gstacks Coverage R Script
	Bwa Alignment Shell Loop
	References
Chapter 8: A Metabarcoding Protocol to Analyze Coastal Planktic Communities Collected by Desalination Plant Filters: From Samp...
	1 Introduction
	2 Materials
		2.1 Subsampling Strategy
		2.2 Bioinformatic Analyses
			2.2.1 Unix Environment
			2.2.2 R Environment (www.r-project.org)
	3 Methods
		3.1 Filters Preparation
			3.1.1 Sampling and Metadata Collection
			3.1.2 Subsampling of the 25 μm Bag Filters
			3.1.3 Preparation of the 25 μm Subsamples for the DNA Extraction
			3.1.4 Subsampling of the 5 μm Cartridge Filters
			3.1.5 Preparation of the 5 μm Subsamples for the DNA Extraction
		3.2 Basic Exploratory Bioinformatic Analyses
			3.2.1 Sequence Analyses of Metabarcoding Samples (Unix and R Commands)
			3.2.2 Exploratory Analyses (R Commands)
	4 Notes
	References
Chapter 9: Barcoding of Antarctic Marine Invertebrates: From Field Sampling to Lab Procedures
	1 Introduction
	2 Materials
		2.1 Sample Collection at  Sea
		2.2 Sorting in the Field
		2.3 Sorting in the  Lab
		2.4 Vouchers Labeling
		2.5 Vouchers Photographic Documentation
		2.6 Subsampling in the  Lab
	3 Methods
		3.1 Sample Collection at  Sea
			3.1.1 Sampling from a  Boat
			3.1.2 SCUBA Sampling
		3.2 Sorting in the Field
		3.3 Sorting in the  Lab
		3.4 Vouchers Labeling
		3.5 Vouchers Photographic Documentation
		3.6 Subsampling in the  Lab
	4 Notes
	References
Chapter 10: DNA Barcoding Procedures for Taxonomical and Phylogenetic Studies in Marine Animals: Porifera as a Case Study
	1 Introduction
	2 Materials
		2.1 Sampling and Preservation
		2.2 Morphological Observations
		2.3 DNA Extraction
		2.4 PCR (Polymerase Chain Reaction) Amplifications and Sequencing
			2.4.1 PCR Reactions
			2.4.2 Agarose Gel Preparation
			2.4.3 PCR Purification
		2.5 Sanger Sequencing
		2.6 Gel Elution (OPTIONAL: See Note 2)
		2.7 Plasmid Cloning (OPTIONAL: See Note 2)
	3 Methods
		3.1 Sample Collection and Preservation
		3.2 Morphological Observations
			3.2.1 Spicule Preparations for Optical Microscope
			3.2.2 Spicule Preparations for Optical Microscope and SEM
			3.2.3 Preparations of Skeleton Organization for Optical Microscopy
			3.2.4 Preparations of Skeleton Organization for SEM
			3.2.5 Preparations for Organic Skeletons (Spongin Fibers)
			3.2.6 Histology for Water-Canal System
		3.3 DNA Extraction
		3.4 PCR (Polymerase Chain Reaction) Amplifications
		3.5 Agarose Gel Electrophoresis
		3.6 Troubleshooting by Gel (See Notes 8 and 9)
		3.7 Troubleshooting by Plasmid Cloning (See Notes 8 and 9)
			3.7.1 Ligation
			3.7.2 Transformation
			3.7.3 Screening and Bacterial Cultivation
			3.7.4 Bacterial DNA Extraction and Sequencing
		3.8 Purification of PCR Products
		3.9 Sanger Sequencing
		3.10 Sequence Processing and Taxonomy Annotation
	4 Notes
	References
Chapter 11: Environmental DNA from Marine Waters and Substrates: Protocols for Sampling and eDNA Extraction
	1 Introduction
	2 Materials
		2.1 Decontamination of Sampling Tools and Workspaces (See Note 1)
		2.2 Surface Marine Water Sampling (See Note 2)
		2.3 Subsurface Marine Water Sampling (See Note 2)
		2.4 Marine Water eDNA Extraction and Quantification Materials
		2.5 Surface Marine Substrate (~ 5 cm) Sampling and Storage (See Note 12)
		2.6 Surface and Subsurface Marine Substrate (Core) Sampling and Storage (See Note 13)
		2.7 Marine Substrate eDNA Extraction and Quantification Materials
	3 Methods
		3.1 Replicates and Controls
		3.2 Decontamination of Sampling Bottles and Other Reusable Equipment (See Note 1)
		3.3 Cleaning and Preparation of Laboratory Workspaces (See Note 1)
		3.4 Surface Marine Water (0-1 M Depth) Sample Collection (See Note 2)
		3.5 Subsurface Marine Water (>1 M Depth) Sample Collection (See Note 2)
		3.6 Marine Water Filtration and Storage
		3.7 Marine Water eDNA Extraction and Quantification
		3.8 Surface Marine Substrate (~5 cm) Sampling and Storage
		3.9 Surface and Subsurface Marine Substrate (Core) Sampling and Storage
		3.10 Marine Substrate eDNA Extraction and Quantification
	4 Notes
	References
Chapter 12: Metataxonomic Analysis of Bacterial Diversity Associated with Marine Organisms
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 Preparation of 0.8% Agarose  Gel
		2.3 Sequencing of 16 rRNA on Illumina MiSeq Platform
		2.4 Taxonomic Classification
	3 Methods
		3.1 DNA Extraction According to the DNeasy PowerSoil Pro Kit (QIAGEN) Protocol
		3.2 Evaluation of DNA Integrity on 0.8% Agarose Gel Electrophoresis
		3.3 Sequencing on Illumina MiSeq Platform
		3.4 Taxonomic Classification
	4 Notes
	References
Chapter 13: From Sequences to Enzymes: Heterologous Expression of Genes from Marine Microbes
	1 Introduction
	2 Materials
		2.1 Making Escherichia coli DH5α and BL21 (DE3) Chemically Competent Cells
		2.2 Molecular Cloning Step
		2.3 Bacterial Transformation
		2.4 Agarose Gel Electrophoresis
		2.5 DNA Plasmid Extraction
		2.6 Protein Expression and Extraction
		2.7 Bradford Assay
		2.8 SDS-Polyacrylamide Electrophoresis (SDS-PAGE)
		2.9 Immunoblotting and Detection
	3 Methods
		3.1 Cloning and Propagation of the Recombinant Expression Vector in Escherichia coli
		3.2 Expression and Extraction of Recombinant Protein
		3.3 Recombinant Protein Detection by SDS-PAGE
		3.4 Recombinant Protein Detection by Western Blotting
	4 Notes
	References
Chapter 14: Expression of Recombinant Cold-Adapted (Hemo)Globins from Marine Bacteria
	1 Introduction
	2 Materials
		2.1 Transformation in the E. coli Strain BL21(DE3)
		2.2 Globin Expression
	3 Methods
		3.1 Transformation in the E. coli Strain BL21(DE3)
		3.2 Globin Expression
	4 Notes
	References
Chapter 15: Isolation of UV-Resistant Marine Bacteria by UV-C Assays
	1 Introduction
	2 Materials
		2.1 Collection of Water/Ice Samples and Surface Sediments
		2.2 Isolation of UV-Resistant Marine Bacteria from Surface Water/Ice/Sediments
		2.3 UV-C Assay
	3 Methods
		3.1 Collection of Surface Water/Ice/Sediments
		3.2 Primary Screening for UV-Resistant Marine Bacteria from Water/Ice Samples
		3.3 Primary Screening for UV-Resistant Marine Bacteria from Surface Sediments
		3.4 Isolation of UV-Resistant Marine Bacteria
		3.5 UV-C Assay
	4 Notes
	References
Chapter 16: Fractionation Protocol of Marine Metabolites
	1 Introduction
	2 Materials
	3 Methods
		3.1 HR-X Fractionation
		3.2 Automated HRX Fractionation on GX-271 ASPEC Gilson Apparatus
	4 Notes
	References
Chapter 17: Detection and Quantification of Small Noncoding RNAs in Marine Diatoms
	1 Introduction
	2 Materials
		2.1 Diatom Culture and Medium
		2.2 RNA Extraction
		2.3 RNA Precipitation to Enrich the Small RNA Fraction (Not Mandatory)
		2.4 Northern Blot Analysis of Low Molecular Weight RNAs
		2.5 Isotope Labeling of Oligo DNA Probes and Washing Buffer (See Note 2)
		2.6 Reverse Transcriptase Reactions
		2.7 Stem-Loop qPCR
	3 Methods
		3.1 Diatom Cultures Conditions
		3.2 RNA Extraction (See Note 4)
		3.3 Northern Blot Analysis
		3.4 Stem-Loop Reversed Transcription Reaction
		3.5 Stem-Loop qPCR
	4 Notes
	References
Chapter 18: Optimized Proteolistic Protocol for the Delivery of the Cas9 Protein in Phaeodactylum tricornutum
	1 Introduction
	2 Materials
		2.1 Culture and Medium
		2.2 gRNA and Cas9 Components
		2.3 Cas9 In Vitro Assay
		2.4 Gold Nanoparticles
		2.5 Gene Gun Equipment
	3 Methods
		3.1 Design of crRNAs
		3.2 crRNA::tracrRNA Duplexes (gRNA)
		3.3 Diatom Culture and Plating
		3.4 Assembly of RNP Complexes
		3.5 Cas9 and gRNAs In Vitro Functional Validation
		3.6 Preparation of Gold Nanoparticles
		3.7 Loading of RNP Complexes onto Gold Particles
		3.8 Proteolistic Shots
		3.9 Replating
	4 Notes
	References
Chapter 19: Production of a Chimeric Mouse-Fish Monoclonal Antibody by the CRISPR/Cas9 Technology
	1 Introduction
	2 Materials
		2.1 Mammalian Cell Culture
		2.2 Construction of CRISPR Plasmid Containing gRNAs and HDR Donor Plasmid
		2.3 Evaluation of Genome Editing in Hybridoma Cells
	3 Methods
		3.1 Design of gRNAs
		3.2 Cloning of gRNAs
		3.3 Gene Knockout
		3.4 Gene Knockin
	4 Notes
	References
Chapter 20: Identification, Characterization, and Expression Analysis of Immunoglobulin Genes from Antarctic Fish by PCR Metho...
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 RNA Extraction
		2.3 cDNA Synthesis
		2.4 5′ and 3′ Rapid Amplification cDNA Ends (RACE)
		2.5 qPCR Analysis*
	3 Methods
		3.1 Identification of the IgH Constant Region Gene in Antarctic Fish
		3.2 5′ RACE of the IgH Constant Region  Gene
		3.3 3′ RACE of IgH Constant Region  Gene
		3.4 Ig Gene Expression Analysis by  qPCR
	4 Notes
	References
Chapter 21: Physical Mapping of Repeated Sequences on Fish Chromosomes by Fluorescence In Situ Hybridization (FISH)
	1 Introduction
	2 Materials
		2.1 Preparation of Chromosome Spreads
		2.2 Preparation of a Labeled Probe
		2.3 Fluorescence In Situ Hybridization (FISH)
	3 Methods
		3.1 Preparation of the Chromosome Spreads on Slides
		3.2 Preparation of Indirectly Labeled DNA Probe
		3.3 FISH
		3.4 Image Analysis
	4 Notes
	References
Chapter 22: Functional Genomics of Fish Erythrocytes
	1 Introduction
	2 Materials
		2.1 Blood Sampling
		2.2 Cell Separation
		2.3 RNA Extraction, Quantity and Quality Determination
		2.4 Determining the Total Transcriptome: RNA Sequencing
		2.5 Picking Genes of Interest: Quantitative PCR
		2.6 Nuclear Run-On Assay Solutions
		2.7 mRNA Stability
	3 Methods
		3.1 Blood Sampling
		3.2 Cell Separation
		3.3 RNA Extraction, Quantity and Quality Determination
		3.4 Determining the Total Transcriptome: RNA Sequencing
		3.5 Picking Genes of Interest: Quantitative PCR
		3.6 Rate of Transcription with Nuclear Run-On Assay
		3.7 mRNA Stability
		3.8 Tying Transcriptional Findings to Protein Function
	4 Notes
	References
Chapter 23: Stain-Free Approach for Western Blot Analysis of Zebrafish Embryos
	1 Introduction
	2 Materials
		2.1 Sample Preparation
		2.2 SDS Stain Free Gel
		2.3 Immunoblotting
		2.4 Software
	3 Methods
		3.1 Sample Preparation
		3.2 Electrophoresis with Stain-Free Gel
		3.3 Protein Transfer and Imaging
		3.4 Antibody Incubation
		3.5 Imaging and Analysis Using the ChemiDoc MP System
		3.6 Validate Total Protein Normalization
	4 Notes
	References
Chapter 24: Proteomics of Fish White Muscle and Western Blotting to Detect Putative Allergens
	1 Introduction
	2 Materials
		2.1 Global Protein Extraction from Fish White Muscle
			2.1.1 Protein Extraction
			2.1.2 Protein Quantification
			2.1.3 1D SDS-PAGE
		2.2 Western Blotting to Detect β-Parvalbumin in the White Muscle Sarcoplasmic Fraction
	3 Methods
		3.1 Global Protein Extraction of Fish White Muscle for Proteomic Analysis
			3.1.1 Protein Extraction
			3.1.2 Protein Quantification
			3.1.3 Proteome Profile Analysis by 1D SDS-PAGE
		3.2 Western Blotting to Detect β-Parvalbumin in a Fish White Muscle Sarcoplasmic Fraction
	4 Notes
	References
Chapter 25: In Vitro Assays for the Bifunctional Acylpeptide Hydrolase (APEH) Enzyme from Antarctic Fish
	1 Introduction
	2 Materials
		2.1 Tissue Collection
		2.2 Total Protein Extraction
		2.3 APEH Exopeptidase Fluorescent Assay
		2.4 Batch-Based Hydrophobic Chromatography of Hemolysates
		2.5 Gel Filtration Chromatography
		2.6 10% Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
		2.7 Oxidation of Bovine Serum Albumin (BSA)
		2.8 Densitometric Analysis
	3 Methods
		3.1 Tissue Collection
		3.2 Exopeptidase Activity
			3.2.1 Preparation of Total Protein Extracts from Fish Blood
			3.2.2 Preparation of Protein Extract from Fish Liver
			3.2.3 Exopeptidase Activity by Spectrofluorometric Assay
		3.3 Endoprotease Assay
			3.3.1 Batch-Based Hydrophobic Interaction Chromatography (HIC)
			3.3.2 Gel Filtration Chromatography on Superdex 200
			3.3.3 10% SDS-PAGE
			3.3.4 BSA Oxidation
			3.3.5 APEH Endoprotease Assay (OPeH Activity)
				Time-Course Incubation
				Densitometric Analysis
	4 Notes
	References
Index