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• Introduction
  • The concept of CLC Single Cell Analysis Module
  • Contact information
  • System requirements and installation
    • System requirements
    • Installation of modules
    • Uninstalling modules
  • Licensing modules
    • Server License
  • The QIAGEN Cell Ontology
  • Reference data management
    • The Reference Data Manager
  • Running tools and workflows
• Data import
  • Import Expression Matrix
  • Import Cell Annotations
  • Import Cell Clusters
  • Import Cell Clonotypes
• Data export
  • Export Cell Ranger HDF5 expression matrix
  • Export Loom expression matrix
  • Export MEX expression matrix
• Creating a count matrix from FASTQ
  • Annotate Reads with Cell and UMI
    • Setting the sample name
    • Interpreting the output of Annotate Reads with Cell and UMI
  • Single Cell RNA-Seq Analysis
    • The Single Cell RNA-Seq Analysis report
    • The Single Cell RNA-Seq Analysis algorithm
• Cell preparation
  • 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
  • Combine Cell Annotations
  • Combine Cell Clusters
  • Convert Metadata to Cell Annotations
  • Update to Common Sample Name
• Noise reduction through feature selection and PCA
  • Feature selection and PCA
    • Calculation of estimated biological variation
• Cell Annotation
  • Predict Cell Types
  • Train Cell Type Classifier
    • Features used for training and prediction
    • Interpreting the output of Train Cell Type Classifier
    • SVMs for cell type classification
  • 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
• Dimensionality reduction
  • Create UMAP Plot
  • Create tSNE Plot
  • Add Information to Plot
• 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
• Immune Repertoire
  • Single Cell Immune Repertoire Analysis
    • The report output from Single Cell Immune Repertoire Analysis
    • The Cell Clonotypes element
    • The clonotype identification algorithm
  • Filter Cell Clonotypes
  • Combine Cell Clonotypes
  • Compare Single Cell Immune Repertoires
    • Interpreting the output of Compare Single Cell Immune Repertoires
  • Convert Clonotypes to Cell Annotations
• Single cell workflows from reads
  • Expression Analysis from Reads
    • Configuring the batch units
    • Output from Expression Analysis from Reads
  • Immune Repertoire Analysis from Reads (10xVDJ)
    • Output from Immune Repertoire Analysis from Reads (10xVDJ)
  • Immune Repertoire and Expression Analysis from Reads (10xVDJ)
    • Configuring the batch units with multiple input types
    • Output from Immune Repertoire and Expression Analysis from Reads (10xVDJ)
• Single cell workflows from imported data
  • Expression Analysis from Matrix
    • Output from Expression Analysis from Matrix
  • Immune Repertoire and Expression Analysis from Clonotypes and Matrix
    • Output from Immune Repertoire and Expression Analysis from Clonotypes and Matrix
• UMAP and tSNE plot functionality
  • Manual Annotation
  • Create Subset
  • Extract to Table
  • Launching of Create Expression Plot
  • Launching of Create Heat Map for Cell Abundance
  • Launching of Differential Expression for Single Cell
• Licensing requirements for the CLC Single Cell Analysis Module
  • Licensing modules on a Workbench
    • Request an evaluation license
    • Download a license using a license order ID
    • Import a license from a file
    • Configure License Server connection
    • Download a static license on a non-networked computer
  • Licensing Server Extensions on a CLC Server
    • Download a static license on a non-networked machine
• Bibliography

Filter Cell Clonotypes

Sometimes it can be desirable to restrict Cell Clonotypes to only a specific subset, for example only productive clonotypes, or only cells that have both the TRA and TRB chains identified. This can be achieved with the Filter Cell Clonotypes tool, which can be found in the Toolbox here:

        Immune Repertoire () | Filter Cell Clonotypes ()

The tool takes a Cell Clonotypes () element as input and produces a filtered Cell Clonotypes () element.

The following options can be adjusted (figure 10.3):

Figure 10.3: The options in the dialog of the Filter Cell Clonotypes tool.

• Barcodes to retain. Multiple elements containing cells can be provided, such as Expression Matrices, Cell Clusters and Cell Annotations. From these, a set of valid cells, identified through the sample and barcode, is obtained as the intersection of the cells in the chosen elements. When used, only the clonotypes for the valid cells are retained in the output.
• Productive. If "Only productive" is enabled, only clonotypes that have a productive CDR3 sequence are retained in the output.
• Barcodes with multiple clonotypes. Barcodes can have more than one clonotype associated with them. Ideally, they contain paired clonotypes for the TRA + TRB chains, or TRG + TRD chains, depending on the cell type. Sometimes a barcode can have fewer or more clonotypes. Configuring the Multiple clonotypes option determines how such barcodes should be handled:
  • Retain all. No filter is applied and all clonotypes are retained.
  • Retain primary / secondary. When a barcode has more clonotypes for the same chain, only the clonotype with the highest number of reads, or second highest, respectively, is retained. Note that Retain primary also retains all the barcodes containing at most one clonotype per chain, while Retain secondary removes them.
  • Retain none. Barcodes containing more than one clonotype for some chain will be removed: only barcodes that have at most one clonotype for each chain are retained.
  Note that the "Retain none" filter option applies at barcode level (the barcode with all its associated clonotypes is removed), while the "Retain primary / secondary" filter option applies at clonotype level.
• Chains to retain. A combination of TRA, TRB, TRG and TRD chains can be chosen and only the clonotypes with the respective chains will be retained. If left empty, no filter is applied.
• Retain only paired clonotypes. Only clonotypes for which a pair is also present are retained. The TRA chain is paired with the TRB chain, and TRG with TRD.

The options above can be mixed and matched to obtain the desired output. Note that the filters are applied in the order given above.

For example, assume we want to only use the primary productive clonotypes with a TRB chain. This can be obtained by enabling "Only productive", setting "Multiple clonotypes" to "Retain primary" and choosing "TRB" in "Chains to retain". If one barcode has two clonotypes with a TRB chain, the primary being non-productive and the secondary being productive, the result will contain the productive chain. Even though this chain was the secondary one in the input, when the tool applies the "Multiple clonotypes" filter, the non-productive clonotype is no longer present in the data and the secondary productive clonotype becomes therefore the primary one.

If a different order for the filters is desired, the tool can be run multiple times with just one of the filter options enabled, in the needed order. For the same example, running the tool first with "Multiple clonotypes" set to "Retain primary" and choosing "TRB" in "Chains to retain", and a second time with "Only productive" enabled, will entirely remove the TRB chains of the mentioned barcode.

The Filter Cell Clonotypes tool can optionally produce a report, summarizing the clonotypes left after filtering. This option is available only when the input element contains just one sample. The output report includes the same information as the report produced by the Single Cell Immune Repertoire Analysis tool, minus the assembly and trimming summaries (see The report output from Single Cell Immune Repertoire Analysis).

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