Haplotyping: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Interrogating the Human Diplome: Computational Methods, Emerging Applications, and Challenges
	1 Introduction
	2 Computational Phasing Methods: A Brief Comparative Overview
		2.1 Statistical Phasing
		2.2 Read-Based Phasing
		2.3 Improving Phasing Methods Via Novel Algorithms
		2.4 Aggregating Phase Information from Different Computational Workflows
		2.5 Tumor Phasing Strategies
		2.6 Annotating Phased Variants in Whole Genomes
	3 Diplomics Phenomena and Applications
		3.1 Two-Hit Models
		3.2 HLA Binding for Neoantigen Discovery
		3.3 Pharmacogenomics
		3.4 Impactful Blood-Brain Barrier Haplotypes
		3.5 Therapeutic Constructs Targeting Specific Sequences
		3.6 Microbiome-Tumor Interactions
		3.7 Alternative Splicing (AS)
		3.8 Phased-Informed Personalized-Pathway Analysis
	4 Validating Diploid-Level Functional Phenomena
	5 Conclusions: The Next Phase in Human Genome Analysis
	References
Chapter 2: Targeted Locus Amplification and Haplotyping
	1 Introduction
	2 Materials
		2.1 Equipment
		2.2 Reagents
	3 Methods
		3.1 Organoid Sample Collection
		3.2 TLA Sample Preparation
			3.2.1 Fixation
			3.2.2 Permeabilization and First Restriction Digestion (RE1)
			3.2.3 Heat Inactivation and Ligation
			3.2.4 Reverse-Crosslinking
			3.2.5 Purification 1
			3.2.6 Second Restriction Digestion (RE2)
			3.2.7 Ligation
			3.2.8 Second Purification
			3.2.9 Quality Control Samples
		3.3 PCR
			3.3.1 Primer Design
			3.3.2 TLA Multiplex PCR
			3.3.3 TLA Multiplex PCR Cleanup
			3.3.4 TLA PCR 2
			3.3.5 TLA PCR Cleanup
		3.4 Sequencing
		3.5 Bioinformatic Analysis and Haplotyping
		3.6 Data Interpretation
	4 Notes
	References
Chapter 3: Full-Length Transcript Phasing with Third-Generation Sequencing
	1 Introduction
	2 Materials
	3 Methods
		3.1 Long-Read Sequencing (LR-Seq) for Haplotype Phasing of Repeat Expansion Disorders
	4 Notes
	References
Chapter 4: Large-Scale Complete Sequencing and Haplotyping of 1-10 kb DNA Molecules Using Short Massively Parallel Reads
	1 Introduction
	2 Materials
		2.1 Plasticware and Instrumentation
		2.2 Reagents
	3 Methods
		3.1 For DNA Molecules Less Than or Equal to 2 kb in Length
			3.1.1 End Repair, A-Tail, and Adapter Ligation
			3.1.2 Circularization
			3.1.3 Rolling Circle Replication (RCR)
			3.1.4 Second Strand Synthesis
		3.2 For DNA Molecules Greater Than 2 kb in Length
			3.2.1 End Repair, A-Tail, and Adapter Ligation
			3.2.2 Primer Extension
			3.2.3 Removing Uracil Bases
			3.2.4 Circularization and Ligation
			3.2.5 Rolling Circle Replication (RCR)
	4 Notes
	References
Chapter 5: Accurate Sequencing and Haplotyping from 10 Cells Using Long Fragment Read (LFR) Technology
	1 Introduction
	2 Materials
		2.1 Plasticware and Instrumentation
		2.2 Reagents
	3 Methods
		3.1 Cell Lysis/Denaturation
		3.2 Multiple Displacement Amplification (MDA)
		3.3 Fragmentation Step 1-Uracil Excision
		3.4 Fragmentation Step 2-Nick Translation
		3.5 Dephosphorylation
		3.6 Barcoded Adapter Ligation
		3.7 Sample Pooling and Purification
		3.8 Primer Extension
		3.9 Second Adapter Ligation
		3.10 PCR Amplification
	4 Notes
	References
Chapter 6: Low-Cost Genome-Scale Phasing with Barcode-Linked Sequencing
	1 Introduction
	2 Materials
		2.1 DNA Extraction
		2.2 DNA Tagmentation
		2.3 Emulsification, PCR, and Emulsion Breakage
		2.4 Sample Purification and Size Selection
		2.5 Sample Enrichment
		2.6 Sample Indexing
		2.7 Sample Purification and Quantification
	3 Methods
		3.1 DNA Extraction
		3.2 DNA Tagmentation
		3.3 Reaction Preparation and Emulsification
		3.4 PCR Amplification
		3.5 Emulsion Breakage
		3.6 Sample Purification and Size Exclusion
		3.7 Sample Enrichment
		3.8 Sample Indexing
		3.9 Sample Purification, Quantification, and Sequencing
		3.10 Data Analysis
	4 Notes
	References
Chapter 7: A Simple Cost-Effective Method for Whole-Genome Sequencing, Haplotyping, and Assembly
	1 Introduction
	2 Materials
		2.1 Plasticware and Instrumentation
		2.2 Reagents
	3 Methods
		3.1 High-Molecular-Weight DNA Isolation from Cells
		3.2 Barcoded Beads
		3.3 Bead Preparation for stLFR
		3.4 stLFR Protocol
		3.5 Analyzing stLFR Data
	4 Notes
	References
Chapter 8: Read-Based Phasing and Analysis of Phased Variants with WhatsHap
	1 Introduction
	2 Installation
	3 Using WhatsHap
		3.1 Preparing Alignment and Variant Files
		3.2 Phasing Diploid Samples
		3.3 Pedigree Phasing
		3.4 Polyploid Phasing
		3.5 Computing Statistics of Phased Variant Files
		3.6 Assigning Reads to Haplotypes and Visualization in IGV
		3.7 Comparing Phased Variant Files
	4 Notes
	References
Chapter 9: HapCUT2: A Method for Phasing Genomes Using Experimental Sequence Data
	1 Introduction
	2 Materials
		2.1 Software Requirements
		2.2 Installation
		2.3 Installation Using Conda
	3 Methods
		3.1 Input Requirements
		3.2 Extraction of Haplotype-Informative Reads
		3.3 Assembly of Haplotypes
		3.4 Output Files
		3.5 Phasing Using Third-Generation Reads
		3.6 Phasing Using Hi-C Reads
		3.7 Phasing Using Linked-Reads (Co-barcoded Reads)
		3.8 Phasing Using Data from Multiple Sequencing Technologies
		3.9 Phasing Indels
		3.10 Parallelizing HapCUT2
	4 Notes
	References
Chapter 10: Determining Complete Chromosomal Haplotypes by mLinker
	1 Introduction
	2 Preparation
		2.1 Installation of mLinker
		2.2 Input Sequencing Data
		2.3 Extract Molecular Linkage
	3 Local Haplotype Inference
		3.1 Haplotype Inference from Linked-Reads/Long-Read Sequencing
		3.2 Determination of High-Confidence Haplotype Blocks
	4 Whole-Chromosome Haplotype Inference
		4.1 Concatenation of Local Haplotype Blocks Using Hi-C Links
		4.2 Verification of the Scaffold Haplotype Solution
		4.3 Calculation of Variant Linkage to the Scaffold Haplotype
		4.4 Final Haplotype Refinement Based on Haplotype Linkage
		4.5 Variant Filtration Based on Haplotype Linkage
	5 Summary
	References
Chapter 11: Haplotyping-Assisted Diploid Assembly and Variant Detection with Linked Reads
	1 Introduction
		1.1 The Development and Evolution of Haplotype Estimation Methods
		1.2 Implementation
	2 Aquila
		2.1 Haplotyping Module
			2.1.1 Contiguity Module
			2.1.2 Local Assembly Module
			2.1.3 Variation Module
		2.2 Related Tools
			2.2.1 Aquila_stLFR
			2.2.2 AquilaSV
			2.2.3 HAPDeNovo
	3 Software Installation and Workflow System
		3.1 Aquila Installation
		3.2 Aquila Workflow
			3.2.1 Haplotyping Virtual DNA Fragments and Partitioning Linked Reads into Phased Small Chunks
			3.2.2 Local Assembly and Constructing Contiguity
			3.2.3 Assembly-Based Variants Calling
			3.2.4 Phasing of All Variants
			3.2.5 Deletion of Unnecessary Intermediate Files
		3.3 The GitHub Links for Installation and Workflow of Other Related Tools
	4 Review of Results
		4.1 Aquila
			4.1.1 Characteristics of Aquila Diploid Assemblies Using 10x Linked-Reads Data
			4.1.2 Assembly-Based Detection and Phasing of SNVs, Small Indels, and SVs
		4.2 AquilaSV: Region-Based Diploid SV Detection from Linked-Reads Data
		4.3 HAPDeNovo: Improving Detection and Phasing for De Novo Mutations from Linked-Reads Data
	5 Discussion
		5.1 Evolution of Sequencing Technologies
		5.2 The Aquila Toolset
		5.3 Future Directions
	References
Chapter 12: Chromosome-Length Haplotypes with StrandPhaseR and Strand-seq
	1 Introduction
	2 Materials
	3 Methods
	4 Notes
	References
Chapter 13: Gamete Binning to Achieve Haplotype-Resolved Genome Assembly
	1 Introduction
	2 Materials
	3 Methods
		3.1 Sequencing Data Required on the Focal Genome
			3.1.1 Long-Read Sequencing
			3.1.2 Single-Cell Sequencing of Gamete Genomes
		3.2 Preparation (See Note 2)
		3.3 Definition of SNP and Deletion Markers (See Note 4)
		3.4 Consensus Calling for Each Gamete Genome
		3.5 Gamete Binning (Phasing Variants Within Contigs)
		3.6 Genetic Mapping (Grouping and Phasing Contigs; See Note 8)
		3.7 Completing the Genetic Map
		3.8 Separation of Long Reads to Haplotype-Specific Chromosomes
		3.9 Independent Assembly with Each Group of Reads (See Notes 5 and 13)
	4 Notes
	References
Chapter 14: Phasing DNA Methylation
	1 Introduction
	2 Materials
		2.1 Nanopore Sequencing Data
		2.2 Required Software Packages
	3 Methods
		3.1 Basecalling
		3.2 Mapping
		3.3 Methylation Calling
			3.3.1 Indexing
			3.3.2 Methylation Calling
		3.4 Variant Calling
		3.5 Phasing of Single Nucleotide Variants
		3.6 Detection of the Methylome Haplotypes
			3.6.1 Preprocessing Methylation Call Data
			3.6.2 Phasing Methylation Data
			3.6.3 Differential Methylation Analysis Between Haplotypes
	4 Notes
		4.1 Methylation Calling
		4.2 Methylation Phasing
	References
Chapter 15: Analysis of 1276 Haplotype-Resolved Genomes Allows Characterization of Cis- and Trans-Abundant Genes
	1 Introduction
		1.1 Moving from a ``World of 1´´ to a ``World of 2´´
		1.2 Many Haplotyping Methods, Not Much Application
		1.3 From Methods to Insight
	2 Key Results
		2.1 Genome-Based Analysis of Cis and Trans Configurations of Coding Variants
			2.1.1 Global Abundance of Cis Configurations
			2.1.2 Significant Cis Abundance in Each Individual Genome
			2.1.3 Significant Cis Abundance Driven by Pairs of Coding Variants
			2.1.4 Expected Versus Observed Cis Fractions
			2.1.5 Pairs of Variants in Cis more Closely Spaced than Pairs in Trans
			2.1.6 Pairs of Variants in Cis More Frequent than Pairs in Trans
		2.2 Gene-Based Analysis of Cis and Trans Configurations of Coding Variants
			2.2.1 A Global Set of Phase-Sensitive Genes
			2.2.2 Classification of Cis- and Trans-Abundant Genes
			2.2.3 Functional Enrichment Differs Between Cis- and Trans-Abundant Genes
			2.2.4 Different Distribution Patterns of Variants in Cross-Validated Cis- and Trans-Abundant Genes
			2.2.5 Cis- and Trans-Abundant Genes Affect Different Parts of the Human Interactome
	3 Materials
		3.1 Data Sets and Phasing Quality
			3.1.1 Haplotype Data from 1092 Genomes Generated by the 1000 Genomes (1000G) Consortium
			3.1.2 Data from 184 Experimentally Phased Genomes from the Personal Genome Project (PGP)
		3.2 Gene Sets
	4 Methods
		4.1 Evaluation of Phasing Quality of Experimentally Haplotype-Resolved Genome Data
		4.2 Annotation of Coding Variants
		4.3 Scoring Cis and Trans Configurations
		4.4 Calculation of Global and Gene-Based Cis/Trans Ratios
			4.4.1 Global Cis/Trans Ratios
			4.4.2 Gene-Based Cis/Trans Ratios
		4.5 Modeling the Expected Composite Probability of a Cis Configuration
		4.6 Simulating Phased Genomes Under Random Assumptions to Derive the Expected Composite Cis/Trans Ratios
		4.7 Gene Set Enrichment and Protein-Protein Interaction Integration with ConsensusPathDB
		4.8 Network Propagation
	5 Conclusions and Outlook
	6 Notes
	References
Chapter 16: Phased Genome Assemblies
	1 Introduction
		1.1 Biotechnology for Genome Assembly and Haplotype Reconstruction
		1.2 Bioinformatic Algorithms for Haploid Genome Assembly
		1.3 Single Individual Haplotyping as a Technique to Perform Haplotype Reconstruction
	2 Phased Genome Assemblies
		2.1 PacBio HiFi as a Base Technology
		2.2 Algorithms for Phased Genome Assemblies
	3 Perspectives
	References
Chapter 17: Haplotype-Assisted Noninvasive Prenatal Diagnosis of Genetic Diseases by Massively Parallel Sequencing of Maternal...
	1 Introduction
	2 Materials
		2.1 Equipment and Consumables
		2.2 Reagents
	3 Methods
		3.1 Plasma cfDNA Sequencing
		3.2 Parental stLFR Sequencing
		3.3 Alignment and Variant Calling
		3.4 Parental Haplotypes Construction
		3.5 Fetal Fraction Estimation
		3.6 Prediction of Fetal Genotype Using Parental Haplotypes (See Note 3)
	4 Notes
	References
Index