Plant Comparative Genomics

Preface
Contents
Contributors
Part I: Phylogenetics and Evolution
	Chapter 1: Orthology Prediction and Phylogenetic Analysis Methods in Plants
		1 Introduction
		2 Materials
			2.1 Equipment
				2.1.1 Hardware
				2.1.2 Software Requirements
					Online Software
				2.1.3 Example  Data
			2.2 Equipment Setup
				2.2.1 Software Installation
		3 Methods
			3.1 Orthology Prediction
				3.1.1 Homology Search
				3.1.2 Orthologs Assignment
				3.1.3 Domain Screening
			3.2 Phylogenetic Analysis
				3.2.1 Multiple Sequence Alignment (MSA)
				3.2.2 Alignments Trimming
				3.2.3 Phylogenetic Inference
					Gene Phylogeny
					Species (Phylogenomic) Phylogeny
		4 Notes
		References
	Chapter 2: Species Tree Inference with SNP Data
		1 Introduction
		2 Materials
			2.1 BEAST 2
			2.2 SNAPP
			2.3 snapp_prep.rb and add_theta_to_log.rb
			2.4 Genotype Data Matrix
			2.5 Species Table
			2.6 Age Constraints
			2.7 Starting Tree
			2.8 Tracer
			2.9 FigTree
		3 Methods
			3.1 Model
			3.2 Generating the XML File with snapp_prep.rb
			3.3 MCMC with BEAST
			3.4 Assessing Stationarity and Convergence with Tracer
			3.5 Obtaining Parameter Estimates with Tracer
			3.6 Generating a Summary Tree with TreeAnnotator
			3.7 Visualizing the Summary Tree in FigTree
		4 Notes
		References
	Chapter 3: High-Throughput Evolutionary Comparative Analysis of Long Intergenic Noncoding RNAs in Multiple Organisms
		1 Introduction
		2 Materials
			2.1 Software and Test/Sample Data
			2.2 Computational Requirements
				2.2.1 Requirements to Run Evolinc-II on the Command Line
				2.2.2 Requirements to Run Evolinc-II in CyVerse´s Discovery Environment
		3 Methods
			3.1 General Overview of Evolinc-II
			3.2 Launching Evolinc-II on the Command Line
			3.3 Launching Evolinc-II in CyVerse´s Discovery Environment
			3.4 Interpreting Results
				3.4.1 Results from a Standard Analysis
				3.4.2 Results from the Phylogenetic and Structural Analyses
		4 Notes
		References
	Chapter 4: NGS-Indel Coder v2.0: A Streamlined Pipeline to Code Indel Characters in Phylogenomic Data
		1 Introduction
		2 Materials
			2.1 System Requirements
			2.2 NGS-Indel Coder Options
			2.3 Input Files Requirements
		3 Methods
			3.1 Running NGS-Indel Coder Including Small Alignment Partitions (Shorter Than Specified Threshold)
				3.1.1 Large Indel Validation and Deletion
				3.1.2 Binary File Creation
				3.1.3 Exon/Intron Identification
			3.2 Running NGS-Indel Coder Omitting Small Alignment Partitions (Shorter Than a Specified Threshold)
				3.2.1 Large Indel Validation and Deletion
				3.2.2 Binary File Creation
				3.2.3 Small Alignment Partition Deletion
				3.2.4 Exon-Intron Identification
		4 Notes
		References
	Chapter 5: An SGSGeneloss-Based Method for Constructing a Gene Presence-Absence Table Using Mosdepth
		1 Introduction
		2 Materials
			2.1 Input Reads
			2.2 Hardware
			2.3 Software
		3 Methods
		4 Notes
		References
	Chapter 6: POInT: A Tool for Modeling Ancient Polyploidies Using Multiple Polyploid Genomes
		1 Introduction
		2 POInT Dataset Assembly/Synteny Block Inference
			2.1 Step 1: Homology Inference
			2.2 Step 2: NCS ``Scaffolding´´
			2.3 Step 3: Pillar Merging and Global Order Inference
		3 Modeling Polyploid Genome Evolution with POInT
		4 Conclusions
		References
Part II: Omics Analysis
	Chapter 7: Searching for Homologous Genes Using Daisychain
		1 Introduction
		2 Materials
			2.1 Software
			2.2 Hardware
		3 Methods
			3.1 Searching for Genes
			3.2 Displaying Gene Homology
			3.3 Displaying Gene Collinearity
		4 Notes
		References
	Chapter 8: Detecting MicroRNAs in Plant Genomes with miRkwood
		1 Introduction
		2 Materials
			2.1 Installation of miRkwood
			2.2 Other Requirements
			2.3 Your  Data
		3 Methods
			3.1 Preparation of Sequencing Reads
			3.2 Running miRkwood
			3.3 Exploring the Results
			3.4 Organization of the Main Results Directory
			3.5 Summary
			3.6 Detailed Results for Known miRNAs
			3.7 Details Results for Novel miRNAs
			3.8 Additional Folders and Files
			3.9 YAML Export
		4 Notes
		References
	Chapter 9: Pangenome Analysis of Plant Transcripts and Coding Sequences
		1 Introduction
			1.1 Background
			1.2 Pangenome History and Concepts
		2 Materials
			2.1 Installation and Up-To-Date Documentation
				2.1.1 Bundled Release
				2.1.2 GitHub Clone/Pull
				2.1.3 Environment and Optional Data (Pfam and SwissProt)
				2.1.4 Docker Container
				2.1.5 High Performance Cluster (HPC) Configuration
		3 Methods
			3.1 Overview of the Pipeline and the get_homologues-est.pl Script
			3.2 Other Scripts
			3.3 Protocol for Plant Transcripts and CDS Sequences
				3.3.1 Preparing Input Sequences, Outgroups and Extracting CDS from Transcripts
				3.3.2 Clustering Sequences
				3.3.3 Annotation of Clusters
				3.3.4 Pangenome Analyses
				3.3.5 Analysis of Accessory Genes/Transcripts
				3.3.6 Downstream Phylogenomic Analyses
		4 Notes
		References
	Chapter 10: Metagenomics Bioinformatic Pipeline
		1 Introduction
		2 Materials
			2.1 Hardware
			2.2 Software
				2.2.1 For Bash
				2.2.2 For R
			2.3 Files
		3 Methods
			3.1 Project Organization and Data Obtainment
			3.2 Assessing Read Quality
			3.3 Trimming and Filtering
			3.4 Metagenome Assembly
			3.5 Metagenome Binning
			3.6 Taxonomic Assignment
			3.7 Diversity Tackled with R
			3.8 A Focused Taxonomic Exploration
		4 Notes
		References
	Chapter 11: Rhizosphere and Endosphere Bacterial Communities Survey by Metagenomics Approach
		1 Introduction
		2 Materials
		3 Methods
			3.1 Collection of Root Endosphere, Rhizosphere, and Bulk Soil Fractions
			3.2 DNA Extraction of Recovered Fractions
				3.2.1 16S rRNA Amplification
			3.3 Bioinformatic Analysis of 16S rRNA Amplicons
				3.3.1 Sequence  Data
				3.3.2 Quality of the Files
				3.3.3 Analysis of 16S rRNA Amplicons with QIIME2
				3.3.4 Importing Raw Sequence (FASTQ) Data into QIIME2
				3.3.5 Multiplexed FASTQ Data with Barcodes in Sequences
				3.3.6 FASTQ Manifest Formats
				3.3.7 Demultiplexing Data (i.e. Mapping Each Sequence to the Sample It Came From)
				3.3.8 Denoising and Selecting Sequences Variants with DADA2 (Length Trimming, Denoising, and Chimera Removal)
				3.3.9 Taxonomic Classification
				3.3.10 Create a Phylogenetic Tree
				3.3.11 Alpha and Beta Diversity Analysis
				3.3.12 Ordination
				3.3.13 Alpha Rarefaction Plotting
		4 Notes
		References
	Chapter 12: Applying Synteny Networks (SynNet) to Study Genomic Arrangements of Protein-Coding Genes in Plants
		1 Introduction
		2 Materials
			2.1 Hardware and System Requirements
			2.2 Software
			2.3 General Guideline to Run the Codes
			2.4 Protocol Starter
		3 Methods
			3.1 Build a SynNet
			3.2 Retrieve the TMBIM Family Network from the SynNet
			3.3 Network Clustering
			3.4 Visualization
				3.4.1 Network Visualization
				3.4.2 Visualizing Clusters in a Phylogenetic Tree with iTOL
				3.4.3 Phylogenomic Profiling
		4 Notes
		References
	Chapter 13: Plant In Situ Hi-C Experimental Protocol and Bioinformatic Analysis
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Labware and Equipment
			2.3 Reagent Setup
		3 Methods
			3.1 Preparation of Plant Material
			3.2 Formaldehyde Cross-Linking of Plant Material
			3.3 Nuclei Isolation
			3.4 Restriction Enzyme Digestion
			3.5 Overhang Fill-in with a Biotinylated Nucleotide
			3.6 In-Situ Ligation of Proximal Ends
			3.7 Cross-Linking Reversal
			3.8 DNA Extraction
			3.9 Biotin Removal from Unligated DNA Ends
			3.10 DNA Shearing
			3.11 Biotin Pulldown
			3.12 Sequencing Library Preparation
			3.13 End Repair and dA-Tailing
			3.14 Adaptor Ligation
			3.15 PCR Amplification (PCR 1)
			3.16 Removal of Adapter Dimers
			3.17 Side qPCR
			3.18 PCR Amplification (PCR 2)
			3.19 Size Selection of Amplified Hi-C Library
			3.20 Library Quality Assessment
			3.21 Sequencing
			3.22 Bioinformatic Analysis
				3.22.1 Software Requirements for Data Analysis
				3.22.2 Read Alignment and Filtering
				3.22.3 Obtain Pairs File from the Bam Generated with HiCUP
				3.22.4 Bin Read Pairs to Obtain a Contact Matrix
				3.22.5 Normalize Matrices to Account for Differences in Sequencing Depth Between Samples
				3.22.6 Correct Matrices to Account for Underlying Biases
				3.22.7 Matrix QC
				3.22.8 Sum Replicate Matrices to Increase the Resolution
				3.22.9 Build a .hic Matrix
				3.22.10 Visualize the Hi-C Matrices
				3.22.11 Identify A/B Compartments
				3.22.12 Identify TADs
				3.22.13 Identify Interaction Peaks
				3.22.14 Identifying Statistically Significant Differential Interactions
		4 Notes
		References
	Chapter 14: Isolation of Boechera stricta Developing Embryos for Hi-C
		1 Introduction
		2 Materials
			2.1 Embryo Fixation, Isolation, and Purification
				2.1.1 Seeds Collection for Isolation of Early-, Middle-, and Late-Stage Embryos
				2.1.2 Formaldehyde Cross-Linking
				2.1.3 Stopping of Cross-Linking
				2.1.4 Seed and Embryo Washing
				2.1.5 Early-Stage Embryo Isolation
		3 Methods
			3.1 Embryo Collection, Fixation, Isolation, and Purification
				3.1.1 Seeds Collection for Isolation of Early-, Middle-, and Late-Stage Embryos
				3.1.2 Formaldehyde Cross-Linking
				3.1.3 Stopping of Cross-Linking
				3.1.4 Sample Washing
				3.1.5 Early-Stage Embryo Isolation and Purification
		4 Notes
		References
Part III: Experimental Procedures for Trait Characterization
	Chapter 15: Discovering the Secrets of Ancient Plants: Recovery of DNA from Museum and Archaeological Plant Specimens
		1 Introduction
		2 Materials
		3 Methods
		4 Notes
		References
	Chapter 16: Use of Allele-Specific Amplification for Rapid Identification of Aromatic and Non-aromatic Rice Germplasms
		1 Introduction
			1.1 Types of Crop Germplasm
		2 Materials
		3 Methods
			3.1 Genomic DNA Isolation Procedure
			3.2 Measurement of DNA Concentration in Spectrophotometer
				3.2.1 Procedure
			3.3 Allele Specific Amplification in PCR
				3.3.1 Procedure
			3.4 Visualization of Amplified Fragments in Agarose Gel Electrophoretic System
				3.4.1 Procedure
		4 Notes
		References
	Chapter 17: Efficient Protein Extraction Protocols for NanoLC-MS/MS Proteomics Analysis of Plant Tissues with High Proteolytic...
		1 Introduction
		2 Materials
			2.1 Total Protein Extraction
			2.2 SDS-Polyacrylamide Gel Electrophoresis
			2.3 Reduction, Alkylation, and Digestion
			2.4 High Reverse-Phase (RP) Fractionation
			2.5 Other Materials and Equipment
		3 Methods
			3.1 Protein Extraction from Tissue
			3.2 Protein Extract Quality Visualization by SDS-PAGE
			3.3 Reduction, Alkylation, and Digestion
			3.4 High pH Reversed-Phase Peptide Fractionation and Enrichment
			3.5 LC/MS-MS Analysis
			3.6 Database Search and Protein/Peptide Identification
		4 Notes
		References
Index