Dedication
Preface
Acknowledgment
Contents
Contributors
Chapter 1: A Predictive Approach to Infer the Activity and Natural Variation of Retrotransposon Families in Plants
1 Introduction
2 Materials
2.1 Genomic Data Retrieval
2.2 De Novo Functional Annotation of the Solanum lycopersicum Genome Using LTRpred
2.3 Distribution of Full-length FIRE Retrotransposons Across the Plant Kingdom
2.4 Concluding Remarks
3 Methods
3.1 De Novo Annotation of Functional Retrotransposons Using LTRpred
3.2 Mobility Assessment of Annotated Retrotransposons Using ALE-Seq
3.3 Assessment of the Natural Variation of Retrotransposon Families in Plants Using metablastr
4 Notes
References
Chapter 2: In Silico Identification of Stress-Associated Transposable Elements in Arabidopsis thaliana Using Public Transcript...
1 Introduction
2 Materials
3 Methods
3.1 Identification of Differentially Expressed Transposable Elements in Stress Conditions
3.2 Identification of Transposable Elements with the Potential to Regulate Genes Differentially Expressed in Stress Conditions
4 Notes
References
Chapter 3: A Practical Guide on Computational Tools and Databases for Transposable Elements in Plants
1 Introduction
2 Materials
2.1 Computational Tools to TE Investigation
2.2 Databases of TEs
3 Methods
3.1 Hands-On in TE Tool and Database
3.1.1 LTR_retriever
3.1.2 Hands-On with HelitronScanner Tool
3.1.3 Hands-On in Novel Database Related to TE
3.2 Data Analysis: What the TE Public Data Is Telling Us By Using Exploratory Analysis
3.3 Conclusion and Future Challenges
4 Notes
References
Chapter 4: A Tutorial of EDTA: Extensive De Novo TE Annotator
1 Introduction
2 Materials
2.1 Overview
2.2 Main Concept
3 Methods
3.1 Getting Started (Installation)
3.1.1 Install Via Conda
3.1.2 Install Via Docker
3.2 Running EDTA
3.3 Output
3.4 Test Run
3.5 Removal of Gene Fragments
4 Notes
References
Chapter 5: Mining of Miniature Transposable Elements in Brassica Species at BrassicaTED
1 Introduction
2 Materials
2.1 Database Structure and Implementation
2.2 Database Contents
3 Methods
3.1 Browse
3.2 Search
3.3 Tools
3.3.1 K BLAST
3.3.2 NCBI BLAST
3.3.3 Sequence Extractor
4 Notes
References
Chapter 6: Genome-Wide Identification of Miniature Inverted-Repeat Transposable Elements by Targeted High-Throughput Sequencing
1 Introduction
2 Materials
2.1 Plant Materials
2.2 Equipment
2.3 Buffers
2.4 Oligonucleotides
2.4.1 Adapters
2.4.2 Primer Sequences
2.4.3 Biotinylated Probes
3 Methods
3.1 Preparation of Solutions Using Ultrapure Water
3.2 DNA Extraction
3.3 Shearing Genomic DNA by Sonication
3.4 End Repair of Fragmented DNA
3.4.1 End-Polishing and 5β² Phosphorylation
3.4.2 Addition of deoxyA to 3β² Termini
3.5 Adapter Ligation
3.5.1 Annealing Adapters
3.5.2 Adapter Ligation of the Prepared DNA Fragments
3.6 To Enrich DNA Fragments of the Correct Size and to Remove Unligated Adapters by Gel Purification
3.7 Enrichment of MITE Flanking Sequences by the First Biotinylated Oligonucleotide Hybridization
3.8 Preparing Dynabeads M-270 Streptavidin
3.9 Binding the Biotin_mJing or DNA Fragment Hybrids to Streptavidin-Dynabeads
3.10 PCR Enrichment of DNA Fragments Before the Second Round of Hybridization
3.10.1 PCR Amplification
3.10.2 PCR Cycling Conditions
3.10.3 Cleaning the PCR Product
3.11 Enrichment of MITE Flanking Sequences by the Second Biotinylated Oligonucleotide Hybridization Step
3.12 Second PCR Enrichment of DNA Fragments
3.12.1 PCR Amplification
3.12.2 PCR Cycling Conditions
3.12.3 Cleaning the PCR Product
3.13 Second Gel Purification
3.14 Determining the DNA Concentration
3.15 Diluting the DNA for Illumina Sequencing
3.16 Sequencing Data Analysis
3.17 Genome-Wide Identification of MITE Insertion Sites
3.18 PCR Validation of mJing Specific and/or Unique Insertions
4 Notes
References
Chapter 7: Identification of Extrachromosomal Circular Forms of Active Transposable Elements Using Mobilome-Seq
1 Introduction
2 Materials
2.1 Genomic DNA Extraction and Purification
2.2 Linear DNA Digestion and Rolling Circle Amplification
2.3 Library Preparation
3 Methods
3.1 Genomic DNA Extraction and Purification
3.2 Linear DNA Digestion and Rolling Circle Amplification
3.3 Library Preparation
4 Notes
References
Chapter 8: Identification of Active Transposable Elements in Plants: The Mobilome-Seq Approach
1 Introduction
2 Materials
2.1 Plant Material and In Vitro Growth Conditions
2.2 Induced Reduction of TE-Silencing
2.3 Sampling and Extraction of DNA
2.4 Mobilome-Sequencing
2.5 Analysis of Mobilome-Seq
2.6 Confirmation of Mobile RetroTEs
3 Methods
3.1 Seed Sterilization, Plating, and Growth
3.2 Sampling of DNA
3.3 Preparation of eccDNA
3.4 Library Preparation and Sequencing
3.5 Analysis of Mobilome-Seq
3.6 Confirmation of RetroTE-Mobility
4 Notes
References
Chapter 9: High-Throughput Profiling of Extrachromosomal Linear DNAs of Long Terminal Repeat Retrotransposons by ALE-seq
1 Introduction
2 Materials
2.1 Oligonucleotides
2.2 Enzymes and Buffers
2.3 Equipment and Consumables
3 Methods
4 Notes
References
Chapter 10: Bioinformatics Analysis Guides to LTR Retrotransposon-Derived Extrachromosomal Linear DNAs Identified by ALE-seq
1 Introduction
2 Materials
2.1 Software Requirement
3 Methods
3.1 Adaptors Trimming (see Note 2)
3.2 Checking the Quality of the Clean Reads
3.3 Reference Index Building and Mapping
3.4 Converting SAM to BAM, Sorting and Indexing of BAM Files (see Note 3)
3.5 Generating Genome Coverage Plot
4 Notes
References
Chapter 11: Determination of TE Insertion Positions Using Transposon Display
1 Introduction
2 Materials
2.1 Plant Material
2.2 Digestion, Purification, and Ligation
2.3 Primers
2.4 PCR
3 Methods
3.1 Preparation of Plant Materials
3.2 Adaptor Preparation
3.3 Digestion and Adaptor Ligation
3.4 PCR
4 Notes
References
Chapter 12: Detection of Transposition Events from Next-Generation Sequencing Data
1 Introduction
2 Materials
2.1 Overview
2.2 Algorithm 1
2.3 Algorithm 2
3 Methods
3.1 Download fastq-dump
3.2 Fetch TIF Program and FASTQ Files for Demonstration
3.3 Download Reference Genome Sequence
3.4 Detection of Low Frequency Transpositions
3.5 Detection of High Frequency Transpositions
3.5.1 Algorithm 1
3.5.2 Algorithm 2
4 Notes
References
Chapter 13: Rapid Detection of Transposon-Induced Genome Rearrangements
1 Introduction
2 Materials
2.1 DNA Isolation
2.2 PCR
2.3 Agarose Gel Electrophoresis
3 Methods
3.1 DNA Extraction
3.2 Polymerase Chain Reaction
3.2.1 Agarose Gel Electrophoresis
3.2.2 High Efficiency Agarose Gel Electrophoresis Method (HEA-GEM)
4 Notes
References
Chapter 14: Detection of Transposable Element Insertions in Arabidopsis Using Sequence Capture
1 Introduction
2 Materials
2.1 Design of TE-Sequence Capture Probes
2.2 DNA Extraction and Library Preparation
2.3 TE-Sequence Capture, Library Amplification, and Sequencing
2.4 Bioinformatic Analysis
2.5 Estimating Sensitivity
3 Methods
3.1 Design of TE-Sequence Capture Probes
3.1.1 Bioinformatic Analysis of Consensus TE Sequences
3.1.2 Population Genomics of TE Insertion Polymorphisms
3.2 DNA Extraction and Library Preparation
3.3 TE-Sequence Capture, Library Amplification, and Sequencing
3.4 Bioinformatic Analysis
3.4.1 Detection of Reads Mapping Partially over TE Sequences
3.4.2 Identification of Potential Novel TE Insertion Sites
3.5 Estimating Sensitivity
3.6 Conclusions
4 Note
References
Chapter 15: Efficient Detection of Transposable Element Insertion Polymorphisms Between Genomes Using Short-Read Sequencing Da...
1 Introduction
2 Materials
2.1 Software
2.1.1 SPLITREADER
2.1.2 TEPID
2.1.3 Other Requirements
2.2 Datasets
2.2.1 Whole-Genome Sequencing Data
2.2.2 Reference Genome Sequence and TE Annotation
3 Methods
3.1 Calling Non-reference TE Presence Variants
3.1.1 Alignment on the Reference Genome Sequence
3.1.2 Calling Non-reference TE Presence Variants from Split- and Discordant Reads
3.1.3 Intersecting and Filtering Non-reference TE Presence Variants by Positive Coverage (DP-filter)
3.1.4 Filtering Non-reference TE Presence Variants by Negative Coverage Drop
3.2 Calling Reference TE Absence Variants
3.3 Using TE-Capture to Estimate the Sensitivity and Specificity of Our TIPs Calling Method
3.3.1 Non-Reference TE Presence Calls
3.3.2 Reference TE Absence Calls
3.4 Conclusion
4 Notes
References
Chapter 16: Quantitative Measurement of Transposon Copy Number Using the Droplet Digital PCR
1 Introduction
2 Materials
2.1 Plant Materials and Growth
2.2 DNA Extraction, Digestion, and Quantitation
2.3 Primers
2.4 Probe-Based ddPCR
3 Methods
3.1 Preparation of Plant Materials
3.2 DNA Extraction, Digestion, and Quantitation
3.3 Probe-Based Droplet Digital PCR
4 Notes
References
Chapter 17: Detecting Signatures of TE Polymorphisms in Short-Read Sequencing Data
1 Introduction
2 Materials
2.1 Reference Genome
2.2 Sequencing Data
2.3 Reference TE Annotation and TE Consensus Sequences
3 Methods
3.1 TE Insertion Polymorphisms
3.2 TE Absence Polymorphisms
3.3 Making Sense of the Output
3.3.1 Visualizing TE Polymorphisms
3.3.2 Combining Variants from Multiple Accessions
3.4 Insights from Long Reads
4 Notes
References
Chapter 18: How to Activate Heat-Responsible Retrotransposon ONSEN in Brassicaceae Species
1 Introduction
2 Materials
3 Methods
3.1 Plant Growth
3.1.1 Chlorine Sterilization
3.1.2 Seeding (Arabidopsis)
3.1.3 Seeding (Others)
3.1.4 Plant Breeding
3.1.5 Heat Treatment
3.1.6 Transfer Plants
3.2 DNA/RNA Extraction
3.3 Real-Time PCR
3.3.1 Quantitative RT-PCR for ONSEN Transcript
3.3.2 Quantitative PCR for Extrachromosomal DNA of ONSEN
3.4 Southern Blotting
4 Notes
References
Chapter 19: Measurement of Genetic Mobility Using a Transposon-Based Marker System in Sorghum
1 Introduction
2 Materials
2.1 DNA Extraction
2.2 PCR for TE-TRAP Marker Generation
2.3 Electrophoresis of Amplicons Using a Fragment Analyzer
2.4 Data Analysis Software
3 Methods
3.1 TEs Sequence Acquisition
3.2 TE-TRAP Primer Design
3.3 DNA Extraction
3.4 TE-TRAP Marker Generation
3.5 Electrophoresis of Amplicons Using a Fragment Analyzer
3.6 TE-TRAP Data Analysis
4 Notes
References
Chapter 20: Sequence-Specific Amplified Polymorphism (SSAP) and Sequence Characterized Amplified Region (SCAR) Markers in Zea ...
1 Introduction
2 Materials
2.1 SSAP Gel Display
2.2 Restriction Digestion and Ligation
2.3 PCR Amplification
2.4 PAGE and Silver Staining
3 Methods
3.1 SSAP Markers
3.1.1 Primer Design
3.1.2 Restriction Digestion and Adaptor Ligation
3.1.3 Pre-selective PCR Amplification
3.1.4 Selective PCR Amplification
3.1.5 Electrophoresis and Visualization of Fragments
3.1.6 Visualization using LI-COR system
3.2 SCAR Marker
3.2.1 Band Elution and Purification of PCR Product
3.2.2 Cloning, Sequencing, BLAST Search and Primer Designing
4 Notes
References
Chapter 21: Analysis of Plant DNA Methylation Profiles Using R
1 Introduction
2 Materials
2.1 Cytosine Methylation Report
2.2 DMRcaller
2.3 Tools to Export DMRs from R
2.4 IGV
3 Methods
3.1 Loading Files
3.2 Calculate Conversion Rate
3.3 Correction for Conversion Rate
3.4 Generate bedGraph for Genome Browser Visualization
3.5 Computing the Methylation Frequency
3.6 Coverage Calculation and Spatial Correlation
3.7 Calculate DNA Methylation in Features
3.8 Call Differentially Methylated Regions (DMRs)
3.9 Call Differentially Methylated Cytosines (DMCs)
3.10 Plot DMRs on Chromosomes
4 Notes
References
Chapter 22: Arabidopsis thaliana Mature Endosperm Dissection and Isolation of Genomic DNA from Mature Seed Tissues
1 Introduction
2 Materials
2.1 Separation of Endosperms and Embryos from Mature Seeds
2.2 Genomic DNA Isolation from Seed Tissues
3 Methods
3.1 Separation of Endosperms and Embryos from Mature Seeds
3.2 Genomic DNA isolation from seed tissues
4 Notes
References
Chapter 23: Transposon-Based Tagging In Silico Using FastPCR Software
1 Introduction
2 Materials
2.1 Software, General Information
2.1.1 Supported Platforms and Dependencies
2.1.2 Downloading and Installing
2.2 The Interface
2.2.1 Inputs to FastPCR
2.2.2 Sequence Entry
2.2.3 Program Output
3 Methods
3.1 In Silico PCR Analysis Steps
3.2 Additional Options Relating to Representation of the Analysis Results
3.3 FastPCR as a Robust Solution for In Silico PCR Assays
3.3.1 Example for Validation of the Software
3.3.2 In Silico PCR Application: An IRAP Example for the Maize Genome
3.3.3 Results Interpretation
4 Notes
References
Chapter 24: Bamboo Transposon Research: Current Status and Perspectives
1 Introduction
2 Evolution and Classical Genetics
3 The Current Status of the Bamboo Genome
4 Bamboo Databases
5 Bamboo Transposons
6 Conclusions
References
Index