Parasite Genomics: Methods and Protocols (Methods in Molecular Biology, 2369)

Preface
Contents
Contributors
Part I: Novel Sequencing and Bioinformatic Pipelines for the Study of Parasite Genomes
	Chapter 1: Nanopore Long Read DNA Sequencing of Protozoan Parasites: Hybrid Genome Assembly of Trypanosoma cruzi
		1 Introduction
		2 Materials
			2.1 Parasites
			2.2 Solutions, Reagents, and Consumables
			2.3 Equipment
		3 Methods
			3.1 High Molecular Weight Genomic DNA Isolation
			3.2 DNA Fragmentation (Optional)
			3.3 ONT Ligation Sequencing Protocol
				3.3.1 DNA Repair and Preparation for Adaptor Ligation
				3.3.2 Barcode Ligation
				3.3.3 Adaptor Ligation
			3.4 ONT Rapid Sequencing Protocol
			3.5 Genome Assembly
				3.5.1 Basecall Reads Using Guppy
				3.5.2 Quality Control of Reads
				3.5.3 Genome Assembly
				3.5.4 Assembly polishing
		4 Notes
		Annex 1 Masurca Configuration  File
		References
	Chapter 2: Chromosomes Conformation Capture Coupled with Next-Generation Sequencing (Hi-C) in Plasmodium falciparum
		1 Introduction
		2 Materials
			2.1 Specific Equipment
			2.2 Kits
			2.3 Reagents and Buffers
		3 Methods
			3.1 Preparation of Crosslinking the parasites (Day 1)
			3.2 Nuclear Extraction and Restriction Digestion (Day 2)
			3.3 Labeling of DNA Ends, Blunt End Ligation, and Crosslinking Reversal (Day 3)
			3.4 DNA Purification and Shearing (Day 4)
			3.5 Pull-Down of Biotinylated  DNA
			3.6 End Repair
			3.7 A-Tailing
			3.8 Adaptor Ligation
			3.9 PCR Amplification and Library Purification
			3.10 Sequencing (Day 5)
			3.11 Read Processing, Normalization, Visualization, and Differential Interaction Analysis
		4 Notes
		References
	Chapter 3: Sequencing and Reconstructing Helminth Mitochondrial Genomes Directly from Genomic Next-Generation Sequencing Data
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
		3 Methods
			3.1 Preparation of Samples for High-Molecular Weight (HMW) Genomic DNA Extraction.
			3.2 Isolation of HMW  gDNA
			3.3 Quantification of gDNA Yield
			3.4 Examination of gDNA Using Agarose Gel Electrophoresis
			3.5 Sequencing
			3.6 Pre-processing of NGS  Data
			3.7 Bioinformatic Assembly, Annotation, and Analysis of Sequence  Data
				3.7.1 Assembly of Sequence  Data
				3.7.2 Evaluation of Mitochondrial Genome Assembly
				3.7.3 Annotation of Sequence  Data
				3.7.4 Additional Analysis of Repeat-Rich Sequences
			3.8 Publication of Annotated mt Genome
		4 Notes
		References
	Chapter 4: Automated Phylogenetic Analysis Using Best Reciprocal BLAST
		1 Introduction
		2 Materials
			2.1 Installation of batch_brb
		3 Methods
			3.1 Setup
			3.2 Data Selection
			3.3 Create BLAST Database
			3.4 Create an Alias Database
			3.5 Retrieve Accessions
			3.6 Identify Putative Orthologs
			3.7 Genome Walk
			3.8 Phylogenetic Trees
			3.9 Analyze Results
			3.10 Finalize and Validate Results
		4 Notes
		References
Part II: Diagnostic Approaches Using Genomic Tools
	Chapter 5: An Illumina MiSeq-Based Amplicon Sequencing Method for the Detection of Mixed Parasite Infections Using the Blastoc...
		1 Introduction
		2 Materials
			2.1 Concentration of Parasite Forms from Feces
			2.2 DNA Extraction
			2.3 Sample Screening and Sequencing Library Preparation
		3 Methods
			3.1 Concentrating Parasite Forms from Feces Via CsCl Centrifugation
			3.2 DNA Extraction
			3.3 Sample Screening and Sequencing Library Preparation
			3.4 Bioinformatic Analysis of Illumina Sequences
		4 Notes
		References
	Chapter 6: Giardia duodenalis: Detection by Quantitative Real-Time PCR and Molecular Diversity
		1 Introduction
		2 Materials
			2.1 Extraction and Purification of Genomic DNA from Stool Samples
			2.2 Molecular Detection of Giardia duodenalis by Quantitative Real-Time PCR (qPCR)
			2.3 Molecular Characterization of Giardia duodenalis
			2.4 Gel Electrophoresis of PCR Products
			2.5 Analysis of DNA Sequences
		3 Methods
			3.1 Extraction and Purification of Genomic DNA from Stool Samples
			3.2 Molecular Detection of Giardia duodenalis by Quantitative Real-Time  PCR
			3.3 Molecular Characterization of Giardia duodenalis
				3.3.1 Amplification of the Glutamate Dehydrogenase Gene of G. duodenalis
				3.3.2 Amplification of the β-Giardin Gene of G. duodenalis
				3.3.3 Amplification of Triosephosphate Isomerase Gene of G. duodenalis
			3.4 Gel Electrophoresis of PCR Products
			3.5 Analysis of DNA Sequences
		4 Notes
		References
Part III: Host-Parasite Interactions: Deciphering Gene Function and Molecular Processes in Parasites
	Chapter 7: Conditional Gene Deletion in Mammalian and Mosquito Stages of Plasmodium berghei Using Dimerizable Cre Recombinase
		1 Introduction
		2 Materials
			2.1 Lab Equipment
			2.2 Plasticware and Glassware
			2.3 Animals, Parasites, Bacteria, and Cells
			2.4 Media, Solutions, and Reagents
				2.4.1 Reagents for DNA Cloning and Genotyping
				2.4.2 Reagents for Cell Culture, Parasite Transfection, and Selection
				2.4.3 Other Reagents
		3 Methods
			3.1 Generation of Plasmids
			3.2 Generation of Conditional Deletion Mutants in Plasmodium berghei
				3.2.1 Plasmid Preparation for Transfection
				3.2.2 Parasite Transfection and Selection (see Note 3)
				3.2.3 Construct Integration Checks
			3.3 Induction of Conditional Gene Deletion
				3.3.1 Conditional Gene Deletion in Asexual Blood Stages
				3.3.2 Conditional Gene Deletion in Sexual Blood Stages Prior to Transmission to Mosquitoes
				3.3.3 Conditional Gene Deletion in Mosquito Stages
				3.3.4 Conditional Gene Deletion in Liver Stages
		4 Notes
		References
	Chapter 8: Coupling Auxin-Inducible Degron System with Ultrastructure Expansion Microscopy to Accelerate the Discovery of Gene...
		1 Introduction
		2 Materials
			2.1 Coverslip Coating
			2.2 Gels Preparation and Expansion
			2.3 Immunostaining
			2.4 Imaging
		3 Methods
			3.1 Coverslips Coating and Preliminary Preparations (Day 0)
			3.2 Extracellular Parasite Preparation (Day 1)
			3.3 Protein Anchoring and Crosslinking Prevention (Day 1)
			3.4 Gelation (Day 1)
			3.5 Denaturation (Day 1)
			3.6 First Round of Expansion (Day 1)
			3.7 Gel Measurement and Shrinkage (Day 2)
			3.8 Immunostaining (Day 2)
			3.9 Final Round of Expansion (Day 2)
			3.10 Imaging (Day 3)
		4 Notes
		References
	Chapter 9: Genome-Wide Analysis of RNA-Protein Interactions in Plasmodium falciparum Using eCLIP-Seq
		1 Introduction
		2 Materials
			2.1 Specific Equipment
			2.2 Kits
			2.3 Specific Consumables
			2.4 Reagents (see Note 1)
			2.5 Primers
			2.6 Recipes
		3 Methods
			3.1 Day 1: Preparation of Parasite Protein Extract and Immunoprecipitation (IP)
				3.1.1 Parasite Extraction (see Note 2)
				3.1.2 UV Crosslinking
				3.1.3 Preparation of Antibody-Coupled Magnetic Beads
				3.1.4 Cell Lysis and Fragmentation
				3.1.5 Immunoprecipitation
			3.2 Day 2: IP Washes, Gel Electrophoresis, and Transfer
				3.2.1 Save Input Samples
				3.2.2 Immunoprecipitation washes
				3.2.3 RNA 5′-End Repair
				3.2.4 RNA 3′-End Repair
				3.2.5 Immunoprecipitation  Wash
				3.2.6 Ligation of RNA Adaptor to IP Samples
				3.2.7 Immunoprecipitation  Wash
				3.2.8 Elution and Denaturation of IP and Input Samples
				3.2.9 Loading SDS-PAGE  Gel
				3.2.10 Transfer to Nitrocellulose Membrane
			3.3 Day 3: Library Preparation
				3.3.1 Size Selection
				3.3.2 RNA Release
				3.3.3 Clean Samples with RNA Clean and Concentrator-5
				3.3.4 5′-End Repair of Input RNA
				3.3.5 3′-End Repair of Input RNA
				3.3.6 Clean Input Samples with RNA Clean and Concentrator-5
				3.3.7 Ligation of RNA Adaptor to Input Samples
				3.3.8 Input Samples Cleanup
				3.3.9 Reverse Transcription of IP and Input Samples
				3.3.10 cDNA Cleanup
				3.3.11 Bead Cleanup
				3.3.12 IP and Input cDNA Ligation
			3.4 Day 4: Library Amplification
				3.4.1 Samples Cleanup
				3.4.2 Quantification by  qPCR
				3.4.3 PCR Amplification
				3.4.4 AMPure Beads Cleanup
				3.4.5 Gel Purification
				3.4.6 Library Quantification and Sequencing
				3.4.7 Read Processing, Normalization, and Visualization
		4 Notes
		References
	Chapter 10: Transcriptional Analysis of Tightly Synchronized Plasmodium falciparum Intraerythrocytic Stages by RT-qPCR
		1 Introduction
		2 Materials
		3 Methods
			3.1 Tight Synchronization of Parasite Cultures
			3.2 Sample Lysis and RNA Collection Using Trizol
			3.3 RNA Extraction and DNase Treatment
				3.3.1 Large RNA Quantity Samples
				3.3.2 Small RNA Quantity Samples
			3.4 Reverse Transcription
			3.5 Analysis of cDNA by Quantitative PCR (qPCR)
			3.6 Data Analysis
				3.6.1 Quality Control
				3.6.2 cDNA Quantification and Normalization
		4 Notes
		References
	Chapter 11: Analysis of the Interaction Between Plasmodium falciparum-Infected Erythrocytes and Human Endothelial Cells Using ...
		1 Introduction
		2 Materials
			2.1 Cells
			2.2 Laminar Flow System
			2.3 Lab Equipment
			2.4 Kits
		3 Methods
			3.1 Co-Incubation of IEs and ECs Using a Laminar Flow System
			3.2 Isolation of RNA/miRNA and NGS Sequencing of  ECs
			3.3 Bioinformatics Data Analysis
			3.4 Imaging
			3.5 R Tracking Script Used for bioinformatics Analysis
		4 Notes
		References
	Chapter 12: Integration of Genomic and Transcriptomic Data to Elucidate Molecular Processes in Babesia divergens
		1 Introduction
		2 Materials
			2.1 B. divergens In Vitro Culture
			2.2 Giemsa Stain
			2.3 B. divergens-Free Merozoite Isolation
			2.4 B. divergens Intraerythrocytic Parasites Isolation
			2.5 RNA Isolation
			2.6 RNAseq Library Preparation
			2.7 Computational Tools
			2.8 Real-Time RT-qPCR
		3 Methods
			3.1 B. divergens In Vitro Culture
			3.2 B. divergens-Free Merozoite Isolation
			3.3 B. divergens Intraerythrocytic Parasites Isolation
			3.4 RNAIsolation
			3.5 RNAseq Library Preparation
			3.6 RNA Sequencing
			3.7 Genome Reassembly, Integration, and Improvement Using Data Obtained from Multiple DNA Sequencing Platforms
			3.8 Differential Expression Analysis Using RNAseq
			3.9 Real-Time RT-qPCR
				3.9.1 Reverse Transcription (RT) Reaction
				3.9.2 Real-Time qPCR
		4 Notes
		References
	Chapter 13: Integration of Functional Genomic, Transcriptomic, and Metabolomic Data to Identify Key Features in Genomic Expres...
		1 Introduction
		2 Materials
			2.1 B. divergens In Vitro Culture
			2.2 CE-TOF/MS
			2.3 GC-QTOF/MS
			2.4 LC-QTOF/MS
			2.5 LC-QqQ/MS
			2.6 Statistics
			2.7 Genomic, Transcriptomic and Metabolomic Data Integration
		3 Methods
			3.1 B. divergens In Vitro Culture
			3.2 B. divergens-Free Merozoite Isolation
			3.3 B. divergens Intraerythrocytic Parasites Isolation
			3.4 Supernatants Isolation from B. divergens In Vitro Cultures
			3.5 Isolation of uRBC and uS
			3.6 Metabolite Extraction
			3.7 CE-TOF/MS Analysis and Data Reprocessing
			3.8 GC-QTOF/MS Analysis and Data Reprocessing
			3.9 LC-QTOF/MS Analysis and Data Reprocessing
			3.10 LC-QqQ/MS Analysis and Data Reprocessing
			3.11 Statistics
			3.12 Biocomputational Integration of Genomic, Transcriptomic, and Metabolomic  Data
		4 Notes
		References
	Chapter 14: Genome Analysis of Programmed DNA Elimination in Parasitic Nematodes
		1 Introduction
		2 Materials
			2.1 Parasitic Nematode Materials
			2.2 Buffers and Solutions
		3 Methods
			3.1 High-Quality Genomic DNA Preparation
			3.2 Genomic Data Analysis on DNA Elimination
				3.2.1 Identify Retained and Eliminated Sequences
				3.2.2 Identify DNA break Regions
				3.2.3 Determine the Consequence of DNA Break Ends in the Somatic Cells
		4 Notes
		References
	Chapter 15: Helminth Microbiota Profiling Using Bacterial 16S rRNA Gene Amplicon Sequencing: From Sampling to Sequence Data Mi...
		1 Introduction
		2 Materials
			2.1 Sampling of Schistosoma mansoni Developmental Stages for Helminth Microbiota Profiling
				2.1.1 Consumables and Equipment
				2.1.2 Buffers, Solutions, Reagents, and Other Compounds
			2.2 DNA Extraction
			2.3 16S rRNA Gene Amplicon Library Preparation and Sequencing on the Illumina MiSeq Platform
				2.3.1 Equipment (see Note 2)
				2.3.2 Reagents and Consumables
			2.4 Analysis of 16S rRNA Gene Sequencing Data Using QIIME2
		3 Methods
			3.1 Sampling of S. mansoni Developmental Stages for Helminth Microbiota Profiling
				3.1.1 Cercariae
				3.1.2 Adult Worms
				3.1.3 Eggs
				3.1.4 Miracidia
				3.1.5 Sporocysts
				3.1.6 Collecting Sample Metadata
			3.2 DNA Extraction
			3.3 16S rRNA Gene Amplicon Library Preparation and Sequencing on the Illumina MiSeq Platform
				3.3.1 Amplicon  PCR
				3.3.2 Amplicon PCR Clean-up
				3.3.3 Index  PCR
				3.3.4 Index PCR Clean-up
				3.3.5 Library Quantification and Pooling
				3.3.6 Library Denaturation and Loading onto the MiSeq
			3.4 Analysis of 16S rRNA Gene Sequencing Data Using QIIME2
			3.5 Sequencing Data Visualization, Statistical Analysis and Mining
		4 Notes
		References
Part IV: Genomics of Parasite-Derived Extracellular Vesicles
	Chapter 16: Methods for the Isolation and Study of Exovesicle DNA from Trypanosomatid Parasites
		1 Introduction
		2 Materials
			2.1 Cell Culture of Epimastigotes and Trypomastigotes Forms of T. cruzi
			2.2 EVs Induction by Serum Starvation
			2.3 Exovesicles Isolation by Ultracentrifugation
			2.4 Exovesicles Isolation by Ultrafiltration
			2.5 Exovesicles DNA Isolation
			2.6 Characterization of the EV-Associated DNA Cargo
		3 Methods
			3.1 In Vitro Differentiation of Epimastigotes into Metacyclic Trypomastigotes
			3.2 Recovery of Trypomastigotes Forms of T. cruzi from Mammalian Cell Cultures and Exovesicles Induction
			3.3 Exovesicles Induction from Epimastigotes Forms of T. cruzi
			3.4 EVs Isolation and Purification
				3.4.1 EVs Isolation by Ultracentrifugation
				3.4.2 EVs Isolation by Ultrafiltration
			3.5 ExoDNA Isolation
			3.6 Characterization of the ExoDNA
		4 Notes
		References
	Chapter 17: Isolation and Analysis of MicroRNAs from Extracellular Vesicles of the Parasitic Model Nematodes Nippostrongylus b...
		1 Introduction
		2 Materials
			2.1 Laboratory Facilities and Equipment
			2.2 General Reagents for Molecular Biology
			2.3 Biological Material: Extracellular Vesicles from Trichuris muris and Nippostrongylus brasiliensis excretory/secretory prod...
			2.4 Kits and Reagents for RNA Extraction and Sequencing
			2.5 Bioinformatic resources
		3 Methods
			3.1 Density Gradient Purification of EVs from Nematodes
			3.2 miRNA Isolation and Quality Control (QC).
			3.3 (Optional) RNA Precipitation Procedure for Small  RNAs
			3.4 miRNA Library Preparation and Sequencing on a NextSeq500 Platform
			3.5 miRNA Identification by Using the miRDeep2 Package
		4 Notes
		References
Index