Table of Contents

• Overview of the repository
  • Data analysed
  • Overview of directories
    • config
    • data
    • environment
    • resources
    • results
    • scripts
• Data access
• Contact
• To Do

pca_visium_analysis

This repository contains code for Visium data processing, downstream analysis, and figure generation for the study titled "Profiling of epithelial functional states and fibroblast phenotypes in hormone therapy-naive localized prostate cancer."

The Visium data was processed using the scanpy package (v1.9.1) and spatial deconvolution was performed using Cell2Location (v0.1.3).

Data analysed:

• In-house Visium samples
  • Batch 1: 2 samples - Visium FFPE V1 (2 x 6.5x6.5mm reactions)
  • Batch 2: 8 samples - Visium FFPE V1 (8 x 6.5x6.5mm reactions)
  • Batch 3: 4 samples - Visium FFPE V2 (2 x 11x11mm reactions)
• Published data from Joakim's lab (PMID 35948798)

Overview of directories:

config/

• Contains parameter files for each script, sample sheet & other ad hoc files

data/

• Contains images, anndata objects (filtered, log_norm, raw, raw_beature_bc_matrix), per-spot histopathology annotation (only for samples in the paper)

environment/

• Contains EDF and ESF files for pca_visium

resources/

• Contains gene lists (e.g., Cepo derived signatures for pnCAFs and Glial cells), and published data (i.e. PMID 35948798)

results/

• Script outputs are saved to corresponding results dir
  • Includes results related to:
    • Basic processing and QC (found in basic_qc/, clustering/, and marker_genes/)
    • Cell2Location analysis (found in 02_cell2location/) - the contents of the subdirectories should be self-explanatory.
      • NOTA BENE - most analyses related to the PCa manuscript can be found here!
    • Cell2Location colocalisation analyses (inhouse and published PMID 35948798 - not relevant to PCa manuscript), H&E plots, and pnCAF and Glial scanpy scores (found in 04_colocalisation_inhouse_and_published)

scripts/:

01_basic_qc/

• Initial pre-processing (without filtering spots), QC & visualisation

02_cell2location/

• 01_h5ad_conversion.R:
  • Convert Seurat object to .h5ad
• 02_train_model.py:
  • Train cell2location model on PCa scRNA-seq reference (cell type minor)
• 03a_cell_type_mapping.py:
  • Map cell types (cell type minor)
• 04_visualize_results.py:
  • Visualize results (cell type minor)
• 05_train_model_major.py:
  • Train cell2location model on PCa scRNA-seq reference (cell type major)
• 06a_cell_type_mapping_major.py:
  • Map cell types (cell type major)
• 07_visualize_results_major.py:
  • Visualize results (cell type major)
• 08_visualise_proportions_minor.R
• 09_visualise_proportions_major.R
• 10_colocalisation_major.R:
  • Summarise cell2location results, & calculate spot-level cell-cell correlations (grouped by sample id & histological feature; cell type major)
• 11_colocalisation_minor.R:
  • Summarise cell2location results, & calculate spot-level cell-cell correlations (grouped by sample id & histological feature; cell type minor)
• 12_train_model_minor_mal.py:
  • Train cell2location model on PCa scRNA-seq reference (cell type minor mal - i.e. a combination of cell type minor annotation and malignant spectrum annotation for Epithelial cells)
• 13a_cell_type_mapping_minor_mal.py:
  • Map cell types (cell type minor mal)
• 14_visualize_results_minor_mal.py:
  • Visualize results (cell type minor mal)
• 15_colocalisation_minor_mal.R:
  • Summarise cell2location results, & calculate spot-level cell-cell correlations (grouped by sample id & histological feature; cell type minor mal)
• 16_train_model_major_temp.py:
  • Train cell2location model on PCa scRNA-seq reference (cell type major temp - i.e. use minor annotation for CAFs and major annotation for all other cell types)
• 17a_cell_type_mapping_major_temp.py:
  • Map cell types (cell type major temp)
• 18_visualize_results_major_temp.py:
  • Visualize results (cell type major temp)
• 19_colocalisation_major_temp.R:
  • Summarise cell2location results, & calculate spot-level cell-cell correlations (grouped by sample id & histological feature; cell type major temp)

Data access

• Processed Visium V1 sequencing and image data is available for in-browser exploration and download through the CELLxGENE portal.
• Raw Visium data will be deposited in the European Genome-Phenome Archive (EGA).

Contact

For further enquires, please either raise an issue via GitHub or email John Reeves (Data Manager - j.reeves(at)garvan.org.au) or Alexander Swarbrick (Lab Head - a.swarbrick(at)garvan.org.au).

To do:

• Further deidentify Cansto IDs - need to be removed from scripts and .dvc files!
• Create a new repo after updates have been made & remove scripts/data not relevant to paper (i.e. analysis of data from PMID 35948798)
• Add CELLxGENE links for V1 (when available)
• Add info on where Visium V2 data can be found
• Add EGA accession number (when available)
• Reference Visium data DVC GitHub repo
• Reference scRNA-seq GitHub repo