Repo for scripts associated with the above thesis by Louise Baldwin
The immune checkpoint inhibitors, or immunotherapies, have been a paradigm shift in cancer treatment. Blocking the immune-inhibitory interactions of CTLA4 with it's ligand B7, or PD1 with it's ligands PDL1/PDL2, can yield remarkable and durable anti-tumour immune reponses in some cancers. However, these therapies are yet to achieve the same outcomes in triple negative breast cancer (TNBC).
As part of my doctoral studies, I utilised single cell sequencing technologies to investigate mechanisms of TNBC immune evasion in two murine models. To analyse both the single cell RNA sequencing and matched single cell VDJ T cell receptor (TCR) sequencing, I generated a number of scripts. The scripts used for the analysis of this dataset can be found in this repo.
The analysis of this project is described below. Each script requires an input file and generates an output file.
| Script | In_file | Results_file |
|---|---|---|
| 1_merge_data.py | Cellranger outputs | updated_merged.h5ad |
| 1a_Scrublet.ipynb | updated_merged.h5ad | merged_scrublet.h5ad |
| 2_QC_clustering.py | merged_scrublet.h5ad | merged_withscrub.h5ad |
| 3_umap.ipynb | merged_withscrub.h5ad | clustered_merged_umap.h5ad |
| 4_major_level_annotation | clustered_merged_umap.h5ad | annotated.h5ad |
| BBKNN_just_Tcells.ipynb | annotated.h5ad | Subset_Tcells_BBKNN.h5ad |
| BBKNN_just_Tcells_annotation.ipynb | Subset_Tcells_BBKNN.h5ad | Subset_Tcells_BBKNN_annotated.h5ad |
| Tcells_mergeVDJ.ipynb | Subset_Tcells_BBKNN_annotated.h5ad | Tcells_withTCR.h5ad |
| TCR_anaysis.ipynb | Tcells_withTCR.h5ad | Plots, figures |
1_merge_data.py: concatenate cellranger outputs into one .adata object
1a_Scrublet.ipynb: predict and remove doublets from the data
2_QC_clustering.py: Run QC, filter and cluster data
3_umap.ipynb: Calculate UMAP
4_major_level_annotation: Annotate clusters at the major (lineage) level
BBKNN_just_Tcells.ipynb: Subset T cells and batch correct using BBKNN
BBKNN_just_Tcells_annotation.ipynb: Annotate T cells at the minor level
Tcells_mergeVDJ.ipynb: Append TCR data to T cell object
TCR_anaysis.ipynb: Carry out TCR QC and analysis
The use of these scripts is described further in the methods (section 2.11), and the outputs are found throughout chapter 5.