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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

The Cell Clonotypes element

A Cell Clonotypes element contains two views, one centered around the identified clonotypes, while the other is centered around the barcodes. Both views contain the following information (see figure 10.2):

Figure 10.2: Views of the same Cell Clonotypes element. Note that not all table columns are shown. Clonotype with number 1 is highlighted in both views. Top: View centered around the identified clonotypes, sorted after the number of barcodes. The paired clonotypes are shown (both TRA + TRB or TRG + TRD chains), and when just one of the chains could be identified, the missing one is listed as NA, as seen for clonotypes 519 and 374. When a clonotype is selected, a second table lists the barcodes with the corresponding clonotype. Bottom: View centered around the barcodes, sorted by barcode. Rows with the same barcode have the same background color when the table is sorted after the barcode.

• Clonotype #. A unique number for the clonotype.
• Chain: Which of the four gene families , , , , the clonotype belongs to. To ease filtering, the families are denoted TRA, TRB, TRG and TRD respectively.
• V / J. The identified V and J reference segment(s), respectively. If a single unambiguous segment cannot be identified, the segments are separated by a comma.
• CDR3 nucleotide sequence. The nucleotide sequence for CDR3 including the V- and J region-encoded conserved motifs.
• CDR3 amino acid sequence. The translated amino acid sequence for the CDR3 nucleotide sequence, provided that it is in-frame.
• CDR3 length. The length of the CDR3 nucleotide sequence.
• Productive. One of three categories are used to characterize the CDR3 nucleotide sequence:
  • Productive. Sequences that are in frame and do not contain a premature stop codon.
  • Out-of-frame. Sequences that have a length that is not a multiple of three.
  • Premature stop codon. Sequences that contain an in-frame premature stop codon.

  Note that the Filter Cell Clonotypes can be used for retaining only the productive clonotypes, see Filter Cell Clonotypes for details.

The view centered around the identified clonotypes additionally contains the numbers of barcodes with the given clonotype. Clicking on a row in this view opens a new table listing the corresponding barcodes (see figure 10.2).

The view centered around the barcodes also provides (see figure 10.2):

• Sample / Barcode. The sample and barcode.
• Reads. The number of reads from the barcode that mapped to the contig from which the specific CDR3 sequence was detected.
• UMIs. The number of unique UMIs the aligned reads correspond to.

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