Step 4. Analysis

Note

This section goes over what is present in STEGO.R. The video tutorial will present some examples of each that will depend on specific biological questions.

Application structure

The analysis section is divided into several sub-sections.

  • Uploaded files

  • Overview

  • TCR -> GEX

  • Automation (TCR -> GEx)

  • GEx -> TCR

Uploaded files

There first tab will show in the side panel the files that can be uploaded.

Main panel

Depending on what you have uploaded, the main panel will render several tables. The first table is of the Seurat object’s meta-data and you will see how it adds the additional labels after the “Sample_Name” column.

Uploaded example of the colitis complication to melanoma therapy dataset (GSE144469)

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Overview

The purpose of this section was to be able to interrogate the overview of the GEx only population dynamics regardless of TCR-seq and visa versa with TCR-seq only.

Main panel (OV)

The GEx section contains the following sections: GEx and TCR

GEx

This section focuses on a general interrogation of the annotations regardless of TCR

  • Percentage

    • This section is divided into percentage (Selected sample vs Colour by:) and can be downloaded as a .csv file.

  • UMAP plot

    • This can be have the full dataset or split by selected samples.

    • Samples can be removed from the “Order of graph (Selected Sample)”

    • Additionally, as there can be overlapping with labels, the user can chose in the “Show all labels?” to show all or selected.

  • Summary chart

    • As we used the semi-supervised annotation stratergy, this usually causes lots of overlap in the UMAP plot.

    • The user can display the “Colour by:” as any of the variables as either a pie chart or bar plot.

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TCR

This section focuses on TCR expansion, and include replicating features from scRepertoire i.e., clonal expansion presented on the UMAP.

  • Overlap

    • Summary table to show the clonal expansion based on the Selected Sample column

    • Upset table that is “V gene with/without CDR3” vs “Selected Sample”

    • If <31 groups to compare, it will render an UPSET plot. This is a type of VENN diagram for more complex comparisons. The top bar represents the unique clones that overlapped the samples if there was a dot and line connection. The right bar graph is the unique clones for each sample

  • Line Graph

    • This section was developed for explore multi-sample/time series based data

    • The way the samples were labelled can split the text by - . etc.

  • Clonal expansion plot

    • bar plot of the clonal

  • Clonality (counts)

    • UMAP plot

Note: Clonal expansion plot and Clonality (counts) variable can be changed with counts (Number_expanded), frequency (Frequency_Expanded) or most abundant clones (Top_clonotypes).

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TCR -> GEx

This section is split into four subsections.

  • Clonal abudance

  • Exapanded

  • ClusTCR

  • Epitope

Clonal abundance

The purpose of the clonal abundance section is for the interrogation of single clones or groups of clones.

The user can select the type of comparison to:

  • Background (BG)

    • This will compare the selected clone(s) to the rest of the data provided.

  • singlets

    • This will compare the selected clone(s) to the cells with a TCR based on the “V gene with/without CDR3” that were only observed once in the data, as these are less likely to be antigen specific.

  • Clones

    • compair to a specific clone(s). This will bring up the ident.2 to select the clones.

In order to render the table efficiently, I have included a filter under the clonal abundance section to the top 50, as well as included a minimum count threshold (default = 2).

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Expansion

This section was aimed to interrogate if there was a consistant gene expression signature of the expanded clones regarless of the TCR arragement.

The sidebar contains some variables to help decide on what the count cut-off could be.

The “Cut off greater than” helps calculate by defaul the D50, or the count above which 50% of the repertiore is represented. This is used to calculate “Cut off greater than” count.

Alternative text I have also used this section in the STEGO.R V3 preprint for compairing the before and after treatment of a single clone.

ClusTCR2

This section will look into the clusters produced in STEP 2.

The user can select which chain they wish to interrogate e.g., TRA, TRB, TRG and TRD, as well as which cluster to interrogate.

The clsuters are ordered in how many total TCR were assocaited with a specific cluster.

The only additional plot that the user can interrogate is the motif.

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Epitope (TCRex)

This epitope section was designed to interrogate the TCRex outputs in STEGO.R.

This section includes in the sidebar to add the TCR headers to the meta-data including the epitope and pathology.

The epitope list is ordered based on TCR with the most sequences.

The top associated epitope for the colitis compliation to melanoma treatment was the melanoma epitope ELAGIGILTV.

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Automation (TCR -> GEX)

  • Clonotype

    • Download public (bar graphs), summary table, dot plot for each public-like clone

    • Download private (single sample)

    • Can restrict based on the calculated priority 1/(sample total * total count)

  • Cluster
    • Download the motif, summary table, dot plot for each public-like and private clusters

      • A common cluster is TRAV1-2 TRAJ33

      • separate alpha and beta cut-offs

    • Priority: 1/(number of nodes * sample total * total count)

  • Epitope/Annotation

    • with the epitopes find the associated epitopes from TCRex

    • Unselect “Add in Epitope data” to focus on the annotations. So, you can identify the TCR linked to specific annotation models (e.g., FunctionTcell)

GEx -> TCR

  • Annotation

    • No longer being developed.

  • Marker

    • Single marker

    • Dual marker