Preface
Contents
Contributors
Chapter 1: The Advent of Precision Immunology: Immunogenetics at the Center of Immune Cell Analysis in Health and Disease
1 Introduction
2 Immunogenetics in the Hematology-Immunology Domain
3 Immunogenetics Methods
4 (Pre- and Post-)Analytical Aspects of Immunogenetics
5 Immunogenetics at the Basis of Precision Immunology
References
Chapter 2: Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma
1 Introduction
1.1 Immunoglobulin Gene Rearrangements
1.2 Clonality Detection in B-Cell Lymphoma Based on BIOMED-2/EuroClonality Assays
1.3 NGS-Based Clonality Detection in B-Cell Lymphomas
1.4 Different NGS Platforms for Clonality Testing: Ion Torrent Versus Illumina
2 Materials
2.1 General Materials and Equipment
2.2 DNA Isolation
2.3 IG-NGS Clonality Assays
2.3.1 Target Amplification and Purifications
2.3.2 Ion Torrent Library Preparation and Sequencing
2.3.3 Illumina Library Preparation and Sequencing
3 Methods
3.1 Samples and Quality Controls
3.2 DNA Isolation
3.2.1 DNA Extraction from Formalin-Fixed Paraffin-Embedded Tissue with Genomic DNA Isolation Kit
3.2.2 DNA Extraction from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue Starting with the Chelex Method
3.2.3 DNA Purification with QIAamp DNA Microcolumn
3.2.4 DNA Extraction from Fresh Frozen Tissue: TSE Method
3.3 Ion Torrent Protocol for IG-NGS Clonality Assessment
3.3.1 Multiplex PCR for Amplification of IGH-FR3, IGHD, and IGK
3.3.2 Cleanup of IGH-FR3, IGHD, and IGK Amplicons
3.3.3 End Repair of Amplicons
3.3.4 Adapter Ligation
3.3.5 Library Amplification
3.3.6 Ion Torrent Sequencing Run
3.4 Illumina Protocol for IG-NGS Clonality Assessment
3.4.1 Multiplex PCR for Amplification of IGH-FR3, IGHD, and IGK
3.4.2 Cleanup of IGH-FR3, IGHD, and IGK Amplicons
3.4.3 Second PCR to Generate Barcoded Amplicons
3.4.4 Cleanup of Barcode-Labeled Amplicons
3.4.5 Illumina Sequencing Run
3.5 Post-Analytical Data Analysis
4 Notes
References
Chapter 3: One-Step Next-Generation Sequencing of Immunoglobulin and T-Cell Receptor Gene Recombinations for MRD Marker Identi...
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 PCR Amplification
2.3 Sample Purification
2.4 Sample Assay
2.5 Pool Sample (2 nM)
2.6 Denaturation Step Before Sequencing
2.7 Sequencing
2.8 Bioinformatic Analysis
3 Methods
3.1 Sample Preparation
3.2 PCR Amplification
3.2.1 Prepare a Mix of Primers for each Target of Interest (See Notes Below)
3.2.2 PCR Amplification
3.3 Sample Purification
3.4 Sample Assay Quantification
3.5 Pool Preparation (2 nM)
3.6 Denaturation Step
3.7 Bioinformatic Analysis with the Vidjil Platform
4 Notes
References
Chapter 4: Immunoglobulin/T-Cell Receptor Gene Rearrangement Analysis Using RNA-Seq
1 Introduction
2 Materials
2.1 RNA-Input Quality Check
2.2 Preparation of RNA-Seq Library
3 Methods
3.1 RNA Isolation and Quality Assessment
3.2 Library Preparation
3.2.1 Purification and Fragmentation of mRNA
3.2.2 First Strand cDNA Synthesis
3.2.3 Second Strand cDNA Synthesis
3.2.4 3β² End Adenylation and Adapter Ligation
3.2.5 DNA Fragment Enrichment
3.2.6 Library Quality Check, Normalization, and Pooling
3.2.7 Sequencing
3.2.8 Raw Data Processing
3.2.9 Quality Control
3.2.10 Adapter Trimming
3.3 Bioinformatic Analysis of the Sequencing Data Using ARResT/Interrogate
4 Notes
References
Chapter 5: Minimal Residual Disease Analysis by Monitoring Immunoglobulin and T-Cell Receptor Gene Rearrangements by Quantitat...
1 Introduction
2 Materials
3 Methods
3.1 ddPCR MRD Quantification for the Target Genes
3.2 DNA Quantification Using the Reference Gene
3.3 ddPCR Results Analysis
3.4 Interpretation of ddPCR MRD Results
3.5 Conclusion
4 Notes
References
Chapter 6: Quality Control for IG/TR Marker Identification and MRD Analysis
1 Introduction
2 IG/TR Marker Identification
2.1 PCR-Based Marker Identification
2.1.1 Control Samples
2.1.2 Distinguishing Between Monoclonal and Polyclonal PCR Products
2.2 NGS-Based Marker Identification
2.2.1 Quality Control of the Library Preparation
2.2.2 Data Analysis
2.3 Choosing Markers for MRD and Optimization of the Clonal-Specific RQ-PCR Systems
3 Interpretation of RQ-PCR MRD Analysis Results
3.1 Identification of False-Positive and False-Negative Results
4 Conclusion
References
Chapter 7: cfDNA-Based NGS IG Analysis in Lymphoma
1 Introduction
2 Materials
2.1 Sample Collection
2.2 cfDNA Extraction
2.3 Digital Droplet PCR (ddPCR)
2.4 One-Step Next-Generation Sequencing (NGS) PCR
2.5 Purification of Subpools by Gel Extraction
3 Methods
3.1 Sample Preparation
3.2 cfDNA Extraction
3.3 Digital Droplet PCR (ddPCR)-Mediated Copy Number Quantification
3.4 NGS Library Preparation
3.5 Next-Generation Sequencing
3.6 Bioinformatic Analysis
4 Notes
References
Chapter 8: Targeted Locus Amplification as Marker Screening Approach to Detect Immunoglobulin (IG) Translocations in B-Cell No...
1 Introduction
2 Materials
2.1 Reagents and Kits
2.2 Instruments and Software
3 Methods
3.1 Mantle Cell Lymphoma and Follicular Lymphoma Cell Collection
3.2 gDNA Extraction and Quality Control
3.3 Targeted Locus Amplification (TLA) Library Preparation
3.3.1 Assembly and Fixation
3.3.2 First Enzymatic Digestion and Ligation
3.3.3 Second Enzymatic Digestion and Ligation
3.3.4 TLA PCR
3.3.5 TLA Library Indexing
3.3.6 Sequencing
3.4 Bioinformatics Analysis
3.5 TLA Sequence Validation by Allele-Specific Oligonucleotide (ASO) Approach
4 Notes
References
Chapter 9: Immunoglobulin/T Cell Receptor Capture Strategy for Comprehensive Immunogenetics
1 Introduction
2 Materials
2.1 DNA Quantification
2.2 DNA Integrity Assessment
2.3 DNA Library Preparation
2.4 DNA Hybridization
2.5 Sequencing of Enriched DNA Library
3 Methods
3.1 Genomic DNA Evaluation and Preparation for DNA Library Generation
3.2 DNA Library Generation
3.3 Quality Control of DNA Libraries
3.4 DNA Hybridization
3.5 Quality Control of Enriched DNA Library
3.6 Sequencing of Enriched DNA Library
3.7 Bioinformatic Analysis of the Sequencing Data Using ARResT/Interrogate
4 Notes
References
Chapter 10: Immunoglobulin Gene Mutational Status Assessment by Next Generation Sequencing in Chronic Lymphocytic Leukemia
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Primer Preparation
2.3 PCR Amplification
2.4 PCR Product Purification
2.5 Quantification of Purified PCR Products
2.6 Library Preparation
2.7 Library Denaturation and Illumina MiSeq Sequencing
2.8 Bioinformatics Analysis
3 Methods
3.1 Template Preparation (See Note 1)
3.1.1 Lymphocyte Isolation from Peripheral Blood Using Density Gradient Separation
3.1.2 Genomic DNA Extraction
3.1.3 RNA Extraction and cDNA Synthesis
3.2 Primer Preparation
3.2.1 Primer Preparation for gDNA Template
3.2.2 Primer Preparation for cDNA Template
3.3 PCR Amplification of IGH Rearrangements
3.4 PCR Product Purification
3.5 Quantification of Purified PCR Products (See Note 7)
3.6 Library Preparation and Quantification
3.7 Library Denaturation and Illumina MiSeq Sequencing
3.8 Bioinformatic Analysis on Vidjil Platform (See Note 9)
4 Notes
References
Chapter 11: NGS-Based B-Cell Receptor Repertoire AnalysisRepertoire analyses in the Context of Inborn Errors of Immunity
1 Introduction
1.1 Selection of the Type of Cell or Tissue
1.2 DNA Versus RNA
1.3 The Number of B Cells that Can Be Studied
1.4 Location of the Primers
1.5 Choosing a Tool to Analyze the Immune Repertoire Data
2 Materials
2.1 Amplification (VH-Cg or VH-Ca from cDNA or VH-JH from DNA)
2.2 Nested PCR
2.3 Merging, Trimming, and Alignment of Reads and Data Analysis
3 Methods
3.1 Amplification of VH-Cg, VH-Ca, or VH-CΞΌ from cDNA
3.2 Amplification of VH-JH from DNA
3.3 Nested PCR and Pooling
3.4 Merging, Trimming, and Alignment of Reads Using Galaxy
3.5 Data Analysis Using the Immune Repertoire Pipeline in Antigen Receptor Galaxy (ARGalaxy) (See Note 13)
3.6 Data Analysis Using the SHM and CSR Tool in ARGalaxy (See Note 13)
4 Notes
References
Chapter 12: Generic Multiplex Digital PCR for Accurate Quantification of T Cells in Copy Number Stable and Unstable DNA Samples
1 Introduction
2 Materials
3 Methods
3.1 Choosing an Experimental Setup
3.2 Multiplex Digital PCR
3.3 Pre-PCR Preparation of Reaction Mixture
3.4 Droplet Generation and PCR Amplification
3.5 Droplet Reading
3.6 Interpretation of Results
3.7 Analysis of Samples with a Lymphoproliferative Component
4 Notes
References
Chapter 13: Gene Engineering T Cells with T-Cell Receptor for Adoptive Therapy
1 Introduction
2 Materials
2.1 Identification of TCR from RNA Isolated from pMHC-Positive T Cells
2.2 Gene Transfer of TCR into T Cells
2.3 In Vitro Validation of TCR
3 Methods
3.1 Identification of TCR from RNA Isolated from pMHC-Positive T Cells
3.1.1 RACE-Ready cDNA, PCR, Cloning, and TCR Sequencing
3.1.2 TCR Sequence Annotation
3.2 Gene Transfer of TCR into T Cells
3.2.1 Packaging TCR Viruses
3.2.2 Activation of Peripheral Blood Mononuclear Cells (PBMCs)
3.2.3 Transduction of PBMCs
3.3 In Vitro Validation of TCR
3.3.1 Surface Expression of TCR Transgene
3.3.2 Sensitivity of TCR Transgene
3.3.3 Specificity of TCR Transgene
3.3.4 Target Cell Recognition
4 Notes
References
Chapter 14: Combined Analysis of Transcriptome and T-Cell Receptor Alpha and Beta (TRA/TRB) Repertoire in Paucicellular Sample...
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Cell Dispensation
2.3 Sequencing
2.4 Analysis
3 Methods
3.1 Sample Preparation
3.2 Cell Dispensation
3.2.1 Dispense Instrument Pre-checks
3.2.2 Staining of Cell Suspension
3.2.3 Dilution and Dispensation of Cells
3.2.4 Imaging of Cells
3.2.5 Analyzing Nanowells (Blank Chip)
3.2.6 Analyzing Nanowells (Printed Chip)
3.3 First and Second Strand cDNA Synthesis
3.4 Cleanup and Concentration After Full-Length cDNA Extraction
3.5 Validation and Quantification of cDNA
3.6 Preparation of TCR a/b Library by Semi-Nested PCR
3.7 Purification of TCR a/b Library
3.8 Validation and Quantification of TCR a/b Library
3.9 Preparation of 5β² differential expression (5β² DE) Library
3.10 Purification of 5β² DE Library
3.11 Validation and Quantification of 5β² DE Library
3.12 Next-Generation Sequencing
3.13 Data Analysis
3.13.1 Primary Analysis of Single-Cell TCR Data
3.13.2 Primary Analysis of RNA-Seq Dat
3.13.3 Downstream Analysis in R
3.13.4 Projecting Gene Expression Data on Cell Coordinates
3.13.5 Projecting VDJ Usage Data on Cell Coordinates
4 Notes
References
Chapter 15: AIRR Community Guide to Planning and Performing AIRR-Seq Experiments
1 Introduction
2 Planning the Experiment
2.1 Organisms
2.2 Samples and Processing
2.3 Bulk vs. Single-Cell Sequencing
2.4 Template Amplification from DNA vs. RNA
2.5 Commercial Kit vs. Homebrew Bulk Methods
2.6 Single Cell: Index Sorting and Bead-Based Emulsion Approaches
2.7 Cost
2.8 Overview of Companion AIRR Community Method Chapters
3 Interpreting the Results
3.1 Overview
3.2 General QC Considerations and Controls
3.3 Clonal Recovery
3.4 PCR Cycle Number
3.5 Sensitivity
3.6 Amplification Bias
3.7 Contamination
3.8 Spurious Amplification Products
3.9 Data Reporting
4 Conclusion
References
Chapter 16: Adaptive Immune Receptor Repertoire (AIRR) Community Guide to TR and IG Gene Annotation
1 Introduction
2 Materials
2.1 Computing Resources
2.2 Software Tools
2.3 Germline Databases
3 Methods
3.1 Preprocessing
3.1.1 Filtering by Sequence or by Clone
3.1.2 Read Length-Related Effects
3.1.3 Productive Vs. Nonproductive Rearrangements
3.2 Gene Annotation
4 Conclusion
References
Chapter 17: Adaptive Immune Receptor Repertoire (AIRR) Community Guide to Repertoire Analysis
1 Introduction
2 Materials
3 Methods
3.1 Gene Usage
3.2 Properties of the CDR3
3.3 Clonal Lineages
3.4 Diversity
3.5 Similarity of AIRR Sequences
3.6 Similarity among Repertoires
3.7 Public Clones
3.8 Detection and Monitoring of Cross-Sample Contamination Events
3.9 B-Cell-Specific Aspects
3.9.1 IG SHM Analysis
3.9.2 Identification of B-Cell Clones
3.9.3 IG Affinity Maturation
3.10 T-Cell-Specific Aspects
4 Conclusion
References
Chapter 18: Bulk gDNA Sequencing of Antibody Heavy-Chain Gene Rearrangements for Detection and Analysis of B-Cell Clone Distri...
1 Introduction
2 Materials
2.1 Primers
2.2 DNA Extraction
2.3 Library Preparation
2.4 Library QC and Sequencing
2.5 Disposables
2.6 Equipment
3 Methods
3.1 Lab Setup
3.2 DNA Purification
3.3 Template Amplification and Initial Quality Control
3.4 Second-Round PCR and Product Purification
3.5 Library Pooling, Purification, and Quantification
3.6 Sequencing
3.7 Software Installation
3.8 Raw Data Processing
3.9 Importing Metadata and Sequence Data into ImmuneDB
3.10 Clonal Inference from Sequencing Data and General Statistics
3.11 Analysis of Clone Numbers and Size Distributions
3.12 Clonal Overlap Analysis
4 Notes
References
Chapter 19: Bulk Sequencing from mRNA with UMI for Evaluation of B-Cell Isotype and Clonal Evolution: A Method by the AIRR Com...
1 Introduction
2 Materials
2.1 General Reagents
2.2 Primers
2.2.1 Human BCR Indexing Primer Set HT for Illumina Sequences
2.2.2 BCR PCR2 Forward Primer i7 HT Index
2.2.3 BCR Indexing Reverse Primer Set HT for Illumina Index Sequences
2.3 Equipment
2.4 Software
3 Methods
3.1 Overview of Wet Bench Protocol
3.2 RNA Extraction
3.3 First-Strand cDNA Synthesis
3.4 First-Round Amplification
3.5 Second-Round PCR Amplification
3.6 Purification of Amplified Libraries
3.7 Library Validation
3.8 Pooling of Samples to Generate Libraries for Sequencing
3.9 Data Analysis Overview
3.10 Raw Data Processing
3.11 Gene Annotation
3.12 Quality Control After Gene Assignment
3.13 Identify Clonally Related Sequences
3.14 Gene Usage by Isotype
3.15 Clonal Lineage Tree Analysis
4 Notes
References
Chapter 20: Single-Cell Analysis and Tracking of Antigen-Specific T Cells: Integrating Paired Chain AIRR-Seq and Transcriptome...
1 Introduction
2 Materials
2.1 10x Genomics Chromium Next GEM Single-Cell V(D)J Kit
2.2 Single-Cell SMART-Seq
2.3 10x Genomics Data Processing and Analysis
2.4 Single-Cell SMART-Seq Data Processing and Analysis
3 Methods
3.1 10x Genomics Chromium Next GEM Single-Cell V(D)J Kit
3.1.1 Coat Tubes for Cell Sort and Count Cells
3.1.2 Load Next GEM Chip G
3.1.3 Post GEM Cleanup and cDNA Amplification
3.1.4 cDNA and Feature Barcode Amplification
3.1.5 Feature Barcode and cDNA Fractionation by Size Selection
3.1.6 Library Construction
Feature Barcode Library Construction by Index-PCR and Purification
Target Enrichment for AIRR Libraries
5β² Gene Expression and AIRR Library Construction: Fragmentation, Adaptor Ligation, and Library Amplification (See Note 13)
3.1.7 Sequencing
3.2 Single-Cell SMART-Seq
3.2.1 Cell Sorting and cDNA Synthesis
Buffer Preparations
Cell Sorting
Preparing Controls
cDNA Synthesis
3.2.2 cDNA Amplification by LD PCR
3.2.3 Purification of Amplified cDNA
3.2.4 Validation Using the Agilent 2100 Bioanalyzer
3.2.5 Library Preparation for Next-Generation Sequencing
Dilute and Prepare cDNA for Tagmentation
Amplify the Tagmented cDNA
Pooling and Purification of Amplified Libraries
3.2.6 Sequencing
3.3 10x Genomics Chromium Next GEM Single-Cell V(D)J Kit Data Processing and Analysis
3.3.1 Setup
3.3.2 Demultiplexing
3.3.3 Alignment
3.3.4 Quality Control
3.3.5 Exploratory Analysis
3.3.6 In-Depth Analysis
3.4 Single-Cell SMART-Seq Data Processing and Analysis
3.4.1 Raw Data Processing
3.4.2 Obtain the Software
3.4.3 Reconstruct TR Sequences with the Assemble Mode
3.4.4 Identify Clonally Related Cells with the Summarise Mode
3.4.5 Quality Control
3.4.6 Interpreting TraCeR Summarise Output
3.4.7 In-Depth Analysis
4 Notes
References
Chapter 21: Quality Control: Chain Pairing Precision and Monitoring of Cross-Sample Contamination: A Method by the AIRR Commun...
1 Introduction
2 Materials
2.1 B-Cell Stimulation to Generate Split-Replicate Cell Samples
2.2 T-Cell Stimulation to Generate Split-Replicate Cell Samples
2.3 Technical Precision Analysis of Paired IG Heavy/Light or Paired TR Alpha/Beta Sequencing
2.4 Laboratory-Scale Global Detection and Monitoring of Cross-Sample Contamination Events
3 Methods
3.1 B-Cell Stimulation for the Generation of Split-Replicate Samples as Repeated Analyses
3.2 T-Cell Stimulation for the Generation of Split-Replicate Samples as Repeated Analyses
3.3 Monitoring the IG Heavy/Light and TR Alpha/Beta Technical Pairing Precision Using Split-Replicate Single-Cell Sequencing S...
3.4 Laboratory-Scale Global Detection and Monitoring of Cross-Sample Contamination Events
4 Notes
References
Chapter 22: Immune Repertoire Analysis on High-Performance Computing Using VDJServer V1: A Method by the AIRR Community
1 Introduction
2 Materials
3 Methods
3.1 Create Project
3.2 Upload Files into Project
3.3 Set File Attributes and Link Paired-End Read Files
3.4 Preprocessing with VDJPipe or pRESTO
3.5 Review Preprocessing Statistics
3.6 Make Job Output Files Available in Project Data Area
3.7 Gene Annotation with IgBLAST
3.8 Define MiAIRR Study Metadata and Repertoire Comparison Groups
3.9 Repertoire Characterization and Comparison with RepCalc
3.10 Visualize Analysis Results and Download Data
4 Notes
References
Chapter 23: Data Sharing and Reuse: A Method by the AIRR Community
1 Introduction
1.1 Experimental Reporting: Minimal Information Standards
1.2 Data Sharing: Data Formats
1.3 Data Sharing: AIRR Data Commons
2 Materials
2.1 Study Design and Reproducibility
2.2 Software Tools
3 Methods
3.1 How to Share AIRR-Seq Data: General Information
3.1.1 Curating Using MiAIRR
3.1.2 Storing AIRR-Compliant Data in INSDC Repositories
3.1.3 Publishing Your Data in the AIRR Data Commons
Collaborating with an Existing ADC Repository Provider
Installing and Running Your Own ADC-Compliant Repository
Implementing the ADC API in an Existing Repository
3.1.4 Sharing Data Through a Non-ADC but AIRR-Compliant Repository
3.2 Finding Data in the AIRR Data Commons
3.2.1 Using the ADC API
3.2.2 Using a Web-Based User Interface
3.3 Methods for Sharing of AIRR-Seq Data
3.3.1 Submission of AIRR-Seq Data to NCBI BioProject, BioSample, and SRA
3.3.2 Publicly Share Data in the VDJServer Community Data Portal
Create Project
Upload Files into Project
Publish Project
3.3.3 Publish the AIRR-Seq Study in the ADC with VDJServer
3.3.4 Install a Local Repository with the iReceptor Turnkey
Download the Software
Install the Software
Loading AIRR-Seq Data
Domain Name, Security, and Public Access to Your Repository
3.4 Methods to Query AIRR-Seq Data in the ADC
3.4.1 Using the UNIX Curl Command
3.4.2 Query ADC API with R
3.4.3 Query ADC API with Python
4 Notes
References
Chapter 24: IMGT Immunoinformatics Tools for Standardized V-DOMAIN Analysis
1 Introduction
2 IMGT Scientific Chart Rules for the Analysis of the V-DOMAIN
2.1 IMGT Gene and Allele Nomenclature and IMGT Reference Directory Sets
2.2 IMGT Unique Numbering for the IG and TR V Domains
2.3 IMGT Standardized Labels and Sequence Description
2.4 IMGT Functionality of IG and TR Genes and Alleles and of Rearranged Sequences
3 IMGT/V-QUEST
3.1 IMGT/V-QUEST Sequence Submission
3.1.1 Your Selection
3.1.2 Sequence Submission
3.1.3 Display Results
3.1.4 Advanced Parameters
3.1.5 Advanced Functionalities
3.2 IMGT/V-QUEST Results for A. Detailed View
3.2.1 Sequence and Result Summary
3.2.2 Detailed Result Sections
3.2.3 Sequence and Result Summary with the Search for Insertions and Deletions in V-REGION
3.2.4 Top of Detailed Results for the Analysis of single chain Fragment variable (scFv)
3.3 IMGT/V-QUEST Results for B. Synthesis View
3.3.1 Summary Table
3.3.2 Detailed Analysis of the JUNCTION
3.3.3 Detailed Result Sections for Alignment of Sequences Expressing the Same V Gene and Allele
3.4 IMGT/V-QUEST Output for Excel File
4 IMGT/HighV-QUEST
4.1 IMGT/HighV-QUEST Sequence Set Submission
4.1.1 The Sequence Submission Form
4.1.2 Display Results
4.1.3 Advanced Parameters
4.1.4 Advanced Functionalities
4.2 IMGT/HighV-QUEST Analysis History Page: Follow-Up and Download of Results
4.3 IMGT/HighV-QUEST Sequence Analysis Results
4.4 IMGT/HighV-QUEST Launch Statistics Page for the Evaluation of IMGT Clonotypes
4.5 IMGT/HighV-QUEST Statistics History Page: Follow-Up and Download of Statistics
4.5.1 Results Sections to be Displayed in the User Web Browser
4.5.2 ``Data´´ Directory
5 IMGT/StatClonotype
5.1 IMGT/StatClonotype Installation and Launch
5.2 IMGT/StatClonotype Uploading of Input Sets of IMGT Clonotypes (AA)
5.3 IMGT/StatClonotype Results
6 IMGT/DomainGapAlign
6.1 IMGT/DomainGapAlign Query and Customization of the Analysis
6.1.1 Standard Parameters and Sequence(s)
6.1.2 Advanced Parameters
6.2 IMGT/DomainGapAlign Results
7 IMGT/Collier-de-Perles
7.1 IMGT/Collier-de-Perles Launched from IMGT Sequence Analysis Tools
7.2 IMGT/Collier-de-Perles Submission Interface
7.3 IMGT/Collier-de-Perles Results
8 Notes
References
25: IMGT/3Dstructure-DB: T-Cell Receptor TR Paratope and Peptide/Major Histocompatibility pMH Contact Sites and Epitope
1 Introduction
2 TR and MH Standardized Description in IMGT/3Dstructure-DB
2.1 TR and MH Chains and Domains
2.1.1 TR Chains and Domains
2.1.2 MH Chains and Domains
2.2 TR V Domains
2.2.1 Definition
2.2.2 IMGT Unique Numbering for V Domain
2.2.3 IMGT Colliers de Perles for V Domain
2.3 MH G Domains
2.3.1 Definition
2.3.2 IMGT Unique Numbering for G Domain
2.3.3 IMGT Colliers de Perles for G Domain
2.4 Residue@ Position and Atom Pair Contacts
3 IMGT pMH Contact Analysis
3.1 IMGT pMH Contact Sites Definition and Determination
3.2 Access to IMGT pMH Contact Sites
3.2.1 IMGT pMH Contact Sites for pMH1
3.2.2 IMGT pMH Contact Sites for pMH2
4 IMGT/3Dstructure-DB Domain Pair Contacts
4.1 IMGT/3DStructure-DB Domain Pair Contacts (Overview)
4.1.1 Domain Pair Contacts for pMH1 Interactions
4.1.2 Domain Pair Contacts for TR/pMH1 Interactions
4.2 IMGT/3Dstructure-DB Domain Pair Contacts (Per Pair)
4.2.1 pMH1 Interactions
4.2.2 TR/pMH1 Interactions
4.3 IMGT Paratope and Epitope
4.4 Bridging IMGT Clonotype (AA), TR-Mimic Antibody and Paratope
4.4.1 IMGT Clonotype (AA) Repertoire and TR Paratope
4.4.2 TR-Mimic Antibody Paratope
5 Availability and Citation
6 Notes
References
Chapter 26: ARResT/Interrogate Immunoprofiling Platform: Concepts, Workflows, and Insights
1 Introduction
1.1 Design
1.2 Primers
1.3 Rearrangements and Junctions
2 Materials
2.1 Sequence Input
2.2 Availability, Requirements, Contact
2.3 Sample Sheet
3 Methods
3.1 A Basic Workflow
3.2 EuroClonality-NGS Assays
4 Notes
References
Chapter 27: Purpose-Built Immunoinformatics for BcR IG/TR Repertoire Data Analysis
1 Introduction
2 Data Processing
2.1 Preprocessing of the Raw Data
2.1.1 Demultiplexing
2.1.2 Adaptor Masking
2.1.3 Format Conversion
2.2 Filtering of the Raw Data
2.3 Synthesis of Paired-End Reads
3 Sequence Annotation with IMGT/HighV-QUEST
4 IMGT/HighV-QUEST Meta-Data Analysis with TRIP
5 High-Throughput Data Analysis with TRIP
5.1 Preselection (Data Curation)
5.2 Selection (Filtering)
5.3 TRIP Analytical Pipeline
5.3.1 Clonotype Computation
5.3.2 Computation of Highly Similar Clonotypes
5.3.3 Repertoire Extraction
5.3.4 CDR3 Length Distribution
5.3.5 pI Distribution
5.3.6 Multiple Value Comparison
5.3.7 Computation of Shared Clonotypes
5.3.8 Repertoire Comparison
5.3.9 Clustering of CDR3 Sequences with Maximum Length Difference of One Amino Acid
5.3.10 Alignment
5.3.11 Insert Identity Groups
5.3.12 Somatic Hypermutation Analysis
5.3.13 Logo Creation
5.3.14 Visualization
5.3.15 Overview
5.3.16 Dependencies
References
Index