Single Cell V(D)J-Seq Analysis
Single Cell V(D)J-Seq Analysis can be found in the Toolbox here:
Toolbox | Single Cell Analysis (
) | Immune Repertoire (
) | Single Cell V(D)J-Seq Analysis (
)
The tool takes as input one or more Sequence Lists (
) of reads that have been annotated using Annotate Single Cell Reads. It outputs a TCR Cell Clonotypes (
) or BCR Cell Clonotypes (
) element (see The Cell Clonotypes element), and optionally a report.
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Sample: All input sequence lists must originate from the same sample, which is set when executing the Annotate Single Cell Reads tool (see Annotate Single Cell Reads). This is because Single Cell V(D)J-Seq Analysis assumes that reads with the same cell barcode that are present in different inputs represent the same cell. The wizard does not allow executing the tool with inputs that are annotated with different samples.
It is important to provide all the data for a sample to Single Cell V(D)J-Seq Analysis at the same time. For example, if one sample was sequenced on 4 lanes of an Illumina sequencer, then all 4 lanes should be supplied together. This allows reads originating from the same cell, but coming from different lanes, to be analyzed jointly and leads to a more accurate clonotype identification. |
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Barcode whitelists: In some protocols, the set of valid barcodes is known in advance, and available as a barcode whitelist. In CLC Single Cell Analysis Module, it is not possible to directly use such a list. Instead, the Filter Cell Clonotypes can be used for filtering the Cell Clonotypes output such that only barcodes that are identified as cells are retained, such as those identified as cells in matched scRNA-Seq data. Additionally, the Filter Cell Clonotypes can be used for retaining only the desired types of clonotypes, for example only those that are productive. See Filter Cell Clonotypes for details. |
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Note: Different runs can result in slightly different results. This is caused by multi-threading of the program combined with the use of probabilistic data structures. The overall content of the Cell Clonotypes should not be markedly different. |
The following options can be adjusted (figure 13.3):
Figure 13.3: The options in the dialog of the Single Cell V(D)J-Seq Analysis tool. Human reference data downloaded from the Reference Data Manager has been selected.
- Reference segments. The V (variable), D (diversity), J (joining) and C (constant) segments. These are used during clonotype identification and determine whether the tool outputs a TCR Cell Clonotypes () or BCR Cell Clonotypes () element. The reference segments can either be
- imported using Import Immune Reference Segments (see Import Immune Reference Segments);
- downloaded from the Reference Data Manager (see Reference data management).
Subsections