This repository contains scripts associated with the pre-print manuscript entitled: Spatial transcriptomics analysis identifies a unique tumor-promoting function of the meningeal stroma in melanoma leptomeningeal disease. The manuscript pre-print can be accessed at bioRxiv or CellPress Sneak Peek. Data used in this code is stored in the Gene Expression Omnibus (GEO) (accession number available upon publication). Whenever possible and/or relevant, intermediate or result files are included in this repository. Unix and R code was contributed by Oscar Ospina.

The .fastq files from Illumina outputs were processed using Space Ranger. The Seurat package was used to import and further process gene expression counts. Biological identification of the spots was achieved using STdeconvolve. Gene expression gradients (STgradient) and spatial gene set enrichment analysis (STenrich) were conducted in spatialGE.

The pre_processing folder contains scripts to use spaceranger mkfastq in order to generate .fastq files from the Illumina outputs and spaceranger count to generate gene expression count matrices.

• pre_processing/visium_mkfastq.sh: A PBS script to run spaceranger mkfastq on an HPC environment and obtain .fastq files from the Illumina sequencing runs. The script was executed separately for each Illumina run. The resulting folder contains demultiplexed .fastq files for each sample.
• pre_processing/visium_count.sh: An example PBS script to run spaceranger count on an HPC environment and obtain gene expression counts. Counts are assigned to genes in the GRCh38-2020-A reference genome (downloadable at https://www.10xgenomics.com/support/software/cell-ranger/downloads/). In order to run the script, a folder containing the tissue images ("HandE_images_spranger_input") taken on each Visium slide area.

The unsupervised_clustering folder contains code to perform unsupervised cluster detection and differential gene expression of the samples (separately and integrated).

• unsupervised_clustering/unsuperv_cluster_merged.Rmd: A pipeline to read Space Ranger outputs and create a Seurat object with gene expression data from all the samples merged in a single matrix. Both log-transformed and SCT-transformed data are generated. Louvain clusters are identified.
• unsupervised_clustering/unsuperv_cluster_separate.Rmd: A pipeline to read Space Ranger outputs and create a Seurat object for each sample, resulting in a named list of seurat objects. Both log-transformed and SCT-transformed data are generated. Louvain clusters are identified for each sample. The named list containing Seurat objects is used in downstream analyses (e.g., spot phenoytyping), and is referenced as '[NAMED_LIST_SEURAT_OBJECTS]' in the scripts.

The spot_phenotyping_stdeconvolve folder contains code to fit Latent Dirichlet Allocation (LDA) models using STdeconvolve. The pipeline involves fitting a series models with varying number of topics, selecting the bets-fitting model, and calculating GSEA scores to assign biological identities to the topics (based on the BluePrint data base). The scripts are run in the following order:

• spot_phenotyping_stdeconvolve/stdeconvolve_model_fitting.Rmd: Code to fit a series of LDA models, one sample at a time.
• spot_phenotyping_stdeconvolve/stdeconvolve_selection_k.Rmd: Code to select the best-fitting model for each sample (i.e., find the most likely number of topics in the sample). The procedure is done one sample at a time.
• spot_phenotyping_stdeconvolve/gsea_topic_deconvolution.Rmd: Code to automatically assign biological identities to the topics using GSEA. The results are manually curated after inspecting log-fold changes of key genes in each topic (see manuscript_figures).

The spatial_gene_set_enrichment folder contains code to conduct spatial gene set enrichment using the STenrich algorithm. Details about the STenrich algorithm can be found here. The code was written to be used in a high performance computing (HPC) environment, however, interested readers can also run the STenrich algorithm using the function implemented in spatialGE.

• spatial_gene_set_enrichment/gene_set_spatial_analysis_hpcrun/run_kegg_pathway_spatial_enrichment_tests_minunits_20_numsds_1.sh: A Slurm script to submit a run of the kegg_pathway_spatial_enrichment_tests.R script to an HPC environment. A similar script is provided to run STenrich using GO gene sets. Note: The STenrich algorithm can also be executed on a regular computer, using spatialGE.
• spatial_gene_set_enrichment/gene_set_spatial_analysis_hpcrun/kegg_pathway_spatial_enrichment_tests.R An R script that executes the STenrich algorithm. A similar script is provided to run STenrich using GO gene sets. Note: The STenrich algorithm can also be executed on a regular computer, using spatialGE.
• spatial_gene_set_enrichment/spatial_plot_geneset_avgexpr.Rmd: An R script to generate plots depicting the average expression of a gene set at each Visium spot.
• spatial_gene_set_enrichment/summarize_stenrich_results.Rmd: Code to generate a summary (heatmap) of the gene sets with evidence of significant spatial enrichment.

The gene_expression_spatial_gradients folder contains code to run the STgradient algorithm. Details about the STgradient algorithm can be found here. The code was written to be used in a high performance computing (HPC) environment, however, interested readers can also run the STgradient algorithm using the function implemented in spatialGE.

• gene_expression_spatial_gradients/compute_pairwise_distances.Rmd: Code to calculate the Euclidean distances of each Visium spot to a reference tissue niche (e.g., tumor, stroma). The distances are used during the execution of the STgradient algorithm.
• gene_expression_spatial_gradients/gene_expr_gradients_hpcrun/run_expr_gradients_correlations.sh: A Slurm script to run the STgradient algorithm in an HPC environment across different combinations of parameters specified in data/stgradient_arguments_runs.txt. Note: The STgradient algorithm can also be executed on a regular computer, using spatialGE.
• gene_expression_spatial_gradients/compute_pairwise_distances.Rmd: The R script containing the STgradient algorithm using an HPC environment. Note: The STgradient algorithm can also be executed on a regular computer, using spatialGE.
• gene_expression_spatial_gradients/compare_correlation_results.Rmd: A script to create a summary (heatmap) of the significant correlations between distance to a tissue niche and gene expression (spatial gradient).

The manuscript_figures folder contains a script to generate figures included in the manuscript

• manuscript_figures/figures_lmm_manuscript.Rmd: An R Markdown script to generate the individual plots in the panel figures of the manuscript.

The data folder contains some of the files necessary to run the spatial analysis pipeline. Other files need to be produced by the user given constraints in size.

• data/loupe_manual_alignments_input: A series of .json files containing coordinates for manual alignment of spots to tissue images. The manual alignment was done only for samples when it was necessary. The files are used during execution of spaceranger count.
• data/deconv_matrices: Matrices containing relative abundance of each LDA topic at each Visium spot
• data/spaceranger_visium_mkfastq.csv: The .csv file used in spaceranger mkfastq, which that relates sample names and Illumina dual-indexes used in library construction.
• data/topic_manual_deconvolution.xlsx: The final biological annotations given to each LDA topic and the original annotation given by GSEA. The rationale for changes in final annotation is also provided in this file.
• data/stgradient_arguments_runs.txt: A file containing the different parameters to run STgradient. Used in the Slurm script: gene_expression_spatial_gradients/gene_expr_gradients_hpcrun/run_expr_gradients_correlations.sh.
• data\spot_domain_majority_classes.xlsx: An workbook containng the labels (tumor|immune|stroma) for each spot based on the STdeconvolve deconvolution. Classes were assigned based on 50% of cell types belonging to one of three categories. This file is used in gene_expression_spatial_gradients/gene_expr_gradients_hpcrun/expr_gradients_stdeconvolve_collapsed.R.