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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

Running tools and workflows

When CLC Single Cell Analysis Module is installed, the toolbox is populated with Single Cell Analysis specific tools and workflows. These include single cell related importers described in Data import and single cell related exporters described in Data export. In addition, the Single Cell Analysis folder will appear in the toolbox as shown in figure 1.1. To launch a tool or workflow either double-click it in the toolbox or use the quick launch browser (figure 1.10). If you are connected to a CLC Server via your Workbench, you will be asked where you would like to run the analysis. We recommend that you run the analysis on a CLC Server when possible.

Figure 1.10: The Launch button is situated in the top left corner of the workbench.

Running tools

All tools require an input. In many cases this will be an imported Expression Matrix, or, when starting from FASTQ, imported reads.

Clicking the Import button in the top toolbar will bring up a list of supported data types, see figure 1.11. Select the appropriate importer and follow the wizard steps. For additional guidance on import settings please refer to the relevant section of Data import. Save the imported data in the navigation area. This is most easily done by choosing to save and selecting a save location. After import, the sample can be found in the navigation area, ready for analysis.

Figure 1.11: Import options with single cell related importers in the lower part. Import Expression Matrix can be expanded to select among the following formats: Cell Ranger HDF5, Loom, MEX, and CSV/TXT. Import Cell Clonotypes can also be expanded to select between the Cell Ranger contig and AIRR formats.

Tools will only run correctly when meaningful input and parameters are provided. If a warning is shown, read it carefully as it will in most cases guide you to the problem.

Select the input based on the description provided in the tool. Although most tools will only allow selection of the correct data type as input, it may still be possible to provide input that does not conform to the tool's expectations. If this happens, the tool will give a warning or gray-out options that cannot be used. The default settings for each tool are suitable for most use cases, but some settings require knowledge of sample preparation or sequencing technology. Most problems arise from inappropriate settings. If a tool produces a report, make sure to inspect it, as it may provide quality control or guidance for how to change settings.

For a full description of running tools, handling results and batch options see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Running_tools_handling_results_batching.html.

Running workflows

CLC Single Cell Analysis Module provides prebuilt workflows for analyzing scRNA-seq data in multiple steps. The workflows can be executed from the toolbox or via the quick launch tool. Input can either be selected from the navigation area or imported using Select files for import. Make sure to select the correct import type. Note: when importing FASTQ, metadata is required to match up read pairs.

Follow the instructions in the wizard steps and save the data by specifying a location in the navigation area. Read more about the provided workflows and how to select proper parameters in section Single cell workflows

For a full description of how to launch workflows and use metadata see https://resources.qiagenbioinformatics.com/manuals/clcgenomicsworkbench/current/index.php?manual=Launching_workflows_individually_in_batches.html.

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