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• Introduction
  • The concept of CLC Single Cell Analysis Module
  • Contact information
  • System requirements
  • Installing modules
    • Licensing modules
    • Uninstalling modules
  • Installing server extensions
    • Licensing server extensions
• Reference data management
  • QIAGEN Sets
• Running tools and workflows
• Data import
  • Import Cell Annotations
  • Import Cell Clusters
  • Import Cell Clonotypes
  • Import Expression Matrix
    • HDF5 formats
    • Other formats
  • Import Peak Count Matrix
  • Import Space Ranger
  • Cell format in importers
  • Import Immune Reference Segments
    • IMSEQ
    • IMGT
    • Output from Import Immune Reference Segments
• Data export
  • Export Cell Ranger HDF5 Expression Matrix
  • Export AnnData Expression Matrix
  • Export h5Seurat Expression Matrix
  • Export Loom Expression Matrix
  • Export MEX Expression Matrix
  • Export TXT Expression Matrix
  • Export Peak Count Matrix
• Prepare Reads
  • Annotate Single Cell Reads
    • Setting the sample name
    • Interpreting the output of Annotate Single Cell Reads
• Creating a Gene Expression Matrix
  • Single Cell RNA-Seq Analysis
    • The Single Cell RNA-Seq Analysis report
    • The Single Cell RNA-Seq Analysis algorithm
  • QC for Single Cell
    • Empty droplets filter
    • Count-based and extra-chromosomal filters
    • Doublets filter
    • Interpreting the output of QC for Single Cell
    • Choosing barcodes to retain
    • The cell calling algorithm
    • The doublet calling algorithm
  • Normalize Single Cell Data
    • When is batch correction appropriate?
    • Interpreting the output of Normalize Single Cell Data
    • The Normalize Single Cell Data algorithm
• Cell Type Classification
  • Browse QIAGEN Cell Ontology
  • Predict Cell Types
    • Interpreting the output of Predict Cell Types
    • Cell type refinement
  • Train Cell Type Classifier
    • Features used for training and prediction
    • Interpreting the output of Train Cell Type Classifier
    • SVMs for cell type classification
  • Update Cell Type Classifier
  • The Cell Type Classifier element
• Expression Analysis
  • Differential Expression for Single Cell
    • Interpreting the output of Differential Expression for Single Cell
    • The differential expression algorithm
  • Create Expression Plot
    • The Heat Map output of Create Expression Plot
    • The Dot Plot output of Create Expression Plot
    • The Violin Plot output of Create Expression Plot
• Velocity Analysis
  • Single Cell Velocity Analysis
    • Interpreting the output of Single Cell Velocity Analysis
    • The velocity estimation algorithm
  • Differential Velocity for Single Cell
  • Score Velocity Genes
    • Interpreting the output of Score Velocity Genes
  • Create Phase Portrait Plot
  • Velocity analysis in workflows
• Spatial Transcriptomics
  • The Spatial Transcriptomics Plot element
• Chromatin Accessibility
  • Single Cell ATAC-Seq Analysis
    • Interpreting the output of Single Cell ATAC-Seq Analysis
    • The report output from Single Cell ATAC-Seq Analysis
    • The Single Cell ATAC-Seq Analysis algorithm
  • Split Read Mapping by Cell
  • Differential Accessibility for Single Cell
    • The differential accessibility algorithm
• Immune Repertoire
  • Single Cell V(D)J-Seq Analysis
    • The report output from Single Cell V(D)J-Seq Analysis
    • The clonotype identification algorithm
  • The Cell Clonotypes element
    • Primary and secondary clonotypes
    • Cell Clonotypes tables
    • Cell Clonotypes alignments
    • Cell Clonotypes Sankey plot
  • Filter Cell Clonotypes
  • Combine Cell Clonotypes
  • Compare Cell Clonotypes
    • Interpreting the output of Compare Cell Clonotypes
  • Convert Clonotypes to Cell Annotations
• Noise reduction through feature selection and dimensionality reduction
  • Feature selection and dimensionality reduction
    • Calculation of estimated biological variation
• Cell Annotation
  • Cluster Single Cell Data
    • The Cluster Single Cell Data algorithm
  • Create Heat Map for Cell Abundance
    • Interpreting the output of Create Heat Map for Cell Abundance
  • Create Cell Annotations from Hashtags
• Dimensionality reduction
  • UMAP for Single Cell
  • tSNE for Single Cell
• Single cell low-dimensional plots functionality
  • Manual annotation
  • Visualizing different types of matrices
  • Create Subset
  • Extract to Table
  • Launching of Create Expression Plot
  • Launching of Create Heat Map for Cell Abundance
  • Launching of Differential Accessibility for Single Cell
  • Launching of Differential Expression for Single Cell
  • Launching of Differential Velocity for Single Cell
  • Launching of Score Velocity Genes
• Utility tools
  • Combine Cell Annotations
  • Update Cell Annotations
  • Convert Metadata to Cell Annotations
  • Combine Cell Clusters
  • Update Cell Clusters
  • Add Information to Plot
  • Update Single Cell Sample Name
• Single cell template workflows from reads
  • Expression Analysis from Reads
    • Configuring the batch units for Expression Analysis from Reads
    • Output from Expression Analysis from Reads
  • Chromatin Accessibility Analysis from Reads
    • Configuring the batch units for Chromatin Accessibility Analysis from Reads
    • Output from Chromatin Accessibility Analysis from Reads
  • Chromatin Accessibility and Expression Analysis from Reads
    • Configuring the batch units for Chromatin Accessibility and Expression Analysis from Reads
    • Output from Chromatin Accessibility and Expression Analysis from Reads
    • Importing reads
  • Immune Repertoire Analysis from Reads (10xV(D)J)
    • Output from Immune Repertoire Analysis from Reads (10xV(D)J)
  • Immune Repertoire and Expression Analysis from Reads (10xV(D)J)
    • Configuring the batch units for Immune Repertoire and Expression Analysis from Reads (10xV(D)J)
    • Output from Immune Repertoire and Expression Analysis from Reads (10xV(D)J)
• Single cell template workflows from imported data
  • Expression Analysis from Matrix
    • Output from Expression Analysis from Matrix
  • Chromatin Accessibility and Expression Analysis from Matrix
    • Output of Chromatin Accessibility and Expression Analysis from Matrix
  • Immune Repertoire and Expression Analysis from Clonotypes and Matrix
    • Output from Immune Repertoire and Expression Analysis from Clonotypes and Matrix
  • Importing data
• Bibliography

Immune Repertoire

Figure 13.1: T and B cell receptors structure. Each pair of chains forms an antigen binding site that binds to specific antigens.

T and B cells form our acquired immune response. They both contain highly variable receptors (see figure 13.1) with binding sites that recognize antigens.

T and B cell receptors (TCR and BCR, respectively) are composed of multiple polypeptide chains: TCRs contain one pair, while BCRs contain two copies of a pair:

• TCR: (TRA) and (TRB), or (TRG) and (TRD).
• BCR: light and heavy. There are two types of light chains in humans: (IGK) and (IGL), while other animals also contain other types of light chains. Once set, the light chain class remains fixed for the life of the B cell. There are five types of heavy chains (IGH) for mammals: , , , and , defining the class of the receptor.

The chains are encoded by genes that undergo somatic recombination. During this process, gene segments are joined with random nucleotides at the junction sites. There are two types of recombination (see figure 13.2):

• VJ recombination, where one V (variable) gene segment is joined to a J (joining) gene segment;
• VDJ recombination, where a D (diversity) gene segment is added between the V and J gene segments.

Figure 13.2: VDJ recombination brings together a V, D, J and C gene segment.

For both types of recombination, a C (constant) gene segment is also added following the J segment.

The TRA and TRG chains are the result of VJ recombination, while the TRB and TRD chains are the result of VDJ recombination. The V, D, J and C gene segments are specific for each TCR chain type.

BCR light chains are the result of VJ recombination, while BCR heavy chains are the result of VDJ recombination. BCR heavy chains have three to four C gene segments. The V, D, J and C gene segments are specific for each BCR light chain type, while they are shared by the BCR heavy chains.

Each chain contains three "complementary-determining regions" (CDRs) (figure 13.2) which form loops in the antigen binding sites. The V(D)J recombination junction is located in the third CDR (CDR3). Due to inclusion of random nucleotides at the junctions between segments, the CDR3 is the most diverse among the three CDRs. Its beginning and end are marked by a conserved cysteine (C) and phenylalanine/tryptophan (F/W) amino-acid in the V and J segments, respectively.

CLC Single Cell Analysis Module offers tools to clonotype reads and characterize the T or B cell receptor repertoire (Single Cell V(D)J-Seq Analysis), filter the repertoires (Filter Cell Clonotypes), combine them across samples (Combine Cell Clonotypes), compare them (Compare Cell Clonotypes) and convert them to cell annotations (Convert Clonotypes to Cell Annotations) for easy visualization on Dimensionality Reduction Plots.

Here, clonotyping consists of identifying which V, D, J and C segments from the reference data (Single Cell V(D)J-Seq Analysis) are used, and extracting the CDR3 region found between the conserved amino acids.

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Subsections

• Single Cell V(D)J-Seq Analysis
• The Cell Clonotypes element
• Filter Cell Clonotypes
• Combine Cell Clonotypes
• Compare Cell Clonotypes
• Convert Clonotypes to Cell Annotations

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