Last updated: 2025-10-02
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Knit directory:
2025_cytoconnect_spatial_workshop/
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Please ensure you follow the instructions below prior to attending the workshop.
You can either create a new R script and copy paste the content of the code blocks or download this Rmd file and run each code block step by step.
Do not just blindly run the code! Make sure you read the description above each of the code block to understand what the code is doing before running them! Sourcing the entire Rmd file will not run anything as every code block has been deliberately annotated such that they won’t execute when the whole file is sourced.
If you have any problems, please reach out to either:
Please install the packages that we will be using in the workshop by running the code block below.
cran_packages <- c(
"BiocManager", "Seurat", "ggplot2", "scales", "patchwork", "qs2",
"viridis", "pak", "here", "hdf5r", "Rfast2"
)
install.packages(cran_packages)
# RCTD only available on Github
pak::pkg_install("dmcable/spacexr")
bioc_packages <- c()
if(length(bioc_packages) > 0) {BiocManager::install(bioc_packages)}Running this chunk will let you know if the packages have been installed properly.
checkSetup <- function() {
library(cli)
cat("\n--------------------------------------\n")
cat(style_bold(col_magenta("\n***Installing General Packages***\n\n")))
not <- c(); not2 <- c()
packages1 <- c(cran_packages, bioc_packages)#, "Test")
for (i in 1:length(packages1)){
if(requireNamespace(packages1[i], quietly = TRUE)==F) {
cat(paste(style_bold(col_red(packages1[i])), "has not been installed\n"))
not <- c(not,i)
} else {
suppressWarnings(suppressMessages(library(as.character(packages1[i]), character.only = TRUE)))
cat(col_yellow(packages1[i]), "is loaded!\n")
}
}
cat("\n--------------------------------------\n")
if (length(not) > 0){
cat(style_bold(bg_red("\n **IMPORTANT** ")),
style_bold(col_yellow("\n\nYou need to install: \n")),
paste(paste(c(packages1[not]), collapse=", ")),
"\n\n--------------------------------------",
"\n\n Use:\n - install.packages(),\n - BiocManager::install() or, \n - use Google to find installation instructions.\n\n", style_bold(col_green("Then run this function again!\n\n")))
} else {
cat("",col_green(style_bold("\n All packages are loaded!\n\n Happy Coding! :)\n\n")))
}
}
checkSetup()The code block below will create a bunch of folders in the location where this script is stored.
# This sets the working directory for all subsequent code chunks to be run
# to the location of this file.
raw_data_dir <- file.path(here::here(), "data", "visium", "raw")
data_dir <- file.path(here::here(), "data", "visium", "data")
if(!dir.exists(raw_data_dir)){dir.create(raw_data_dir, recursive = TRUE)}
if(!dir.exists(data_dir)){dir.create(data_dir, recursive = TRUE)}Please source the code block below to load a function to check the integrity of the downloaded files.
check_md5 <- function(files_expected_md5sum) {
# files_expected_md5sum must be a named vector where the name is the path to the file
# and the value is the expected md5sum
library(cli)
library(tools)
error <- combine_ansi_styles("red", "bold")
need_redownload <- FALSE
for (filename in names(files_expected_md5sum)) {
actual_md5sum <- md5sum(filename)
expected_md5sum <- files_expected_md5sum[filename]
if (actual_md5sum != expected_md5sum) {
cat(error(paste(
filename, "is corrupted. Actual md5sum:", actual_md5sum, "!= Expected md5sum:", expected_md5sum, "\n"
)
))
need_redownload <- TRUE
}
}
if (need_redownload) {
cat(error("Some files are corrupted. Please redownload"))
}
}Note: if you encounter a timeout error
(after 60 seconds) while downloading the files, you can increase the
timeout to say 6,000 seconds to give R more time to download the files
by running
options(timeout = max(6000, getOption("timeout")))
The code block below will download the count matrix and spatial information required to load the Visium data in.
download.file("https://cf.10xgenomics.com/samples/spatial-exp/3.0.0/Visium_V2_Human_Colon_Cancer_P2/Visium_V2_Human_Colon_Cancer_P2_filtered_feature_bc_matrix.h5",
destfile = file.path(raw_data_dir, "Visium_V2_Human_Colon_Cancer_P2_filtered_feature_bc_matrix.h5"),
method = "curl")
download.file("https://cf.10xgenomics.com/samples/spatial-exp/3.0.0/Visium_V2_Human_Colon_Cancer_P2/Visium_V2_Human_Colon_Cancer_P2_spatial.tar.gz",
destfile = file.path(raw_data_dir, "spatial.tar.gz"),
method = "curl")The code below will check the integrity of the downloaded files. If you get any red warning, please re-download the files that were found to be corrupted using the code block above.
# check md5sum of the downloaded files.
md5sum_str <- c(
"0a9733cf0dfd3dfaa3b6c1c5c24bcbed",
"a32edc825ab7089fca7848fc80cd5113"
)
names(md5sum_str) <- c(
file.path(raw_data_dir, "Visium_V2_Human_Colon_Cancer_P2_filtered_feature_bc_matrix.h5"),
file.path(raw_data_dir, "spatial.tar.gz")
)
check_md5(md5sum_str)If there are no warning from the code block above, proceed to untar
the spatial.tar.gz file using the code block below.
untar(file.path(raw_data_dir, "spatial.tar.gz"), exdir = raw_data_dir)TODO. Need to upload the qs2 file somewhere they can download.
The following file is optional and is only required if you would like to try out the demo we will be showing on how to use loupe browser to make some QC step simpler.
download.file(
"https://cf.10xgenomics.com/samples/spatial-exp/3.0.0/Visium_V2_Human_Colon_Cancer_P2/Visium_V2_Human_Colon_Cancer_P2_cloupe.cloupe",
destfile = file.path(raw_data_dir, "Visium_V2_Human_Colon_Cancer_P2_cloupe.cloupe"),
method = "curl"
)
md5sum_str <- c("c5726e203c21a2fc3432831ea7c74252")
names(md5sum_str) <- c(
file.path(raw_data_dir, "Visium_V2_Human_Colon_Cancer_P2_cloupe.cloupe")
)
check_md5(md5sum_str)Install Loupe Browser at this link: https://www.10xgenomics.com/support/software/loupe-browser/latest
sessionInfo()R version 4.5.1 (2025-06-13)
Platform: aarch64-apple-darwin20
Running under: macOS Sequoia 15.5
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/4.5-arm64/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.5-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.1
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
time zone: Australia/Melbourne
tzcode source: internal
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] workflowr_1.7.2
loaded via a namespace (and not attached):
[1] vctrs_0.6.5 httr_1.4.7 cli_3.6.5 knitr_1.50
[5] rlang_1.1.6 xfun_0.53 stringi_1.8.7 processx_3.8.6
[9] promises_1.3.3 jsonlite_2.0.0 glue_1.8.0 rprojroot_2.1.1
[13] git2r_0.36.2 htmltools_0.5.8.1 httpuv_1.6.16 ps_1.9.1
[17] sass_0.4.10 rmarkdown_2.29 jquerylib_0.1.4 tibble_3.3.0
[21] evaluate_1.0.5 fastmap_1.2.0 yaml_2.3.10 lifecycle_1.0.4
[25] whisker_0.4.1 stringr_1.5.2 compiler_4.5.1 fs_1.6.6
[29] pkgconfig_2.0.3 Rcpp_1.1.0 rstudioapi_0.17.1 later_1.4.4
[33] digest_0.6.37 R6_2.6.1 pillar_1.11.0 callr_3.7.6
[37] magrittr_2.0.4 bslib_0.9.0 tools_4.5.1 cachem_1.1.0
[41] getPass_0.2-4
sessionInfo()R version 4.5.1 (2025-06-13)
Platform: aarch64-apple-darwin20
Running under: macOS Sequoia 15.5
Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/4.5-arm64/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.5-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.1
locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
time zone: Australia/Melbourne
tzcode source: internal
attached base packages:
[1] stats graphics grDevices utils datasets methods base
other attached packages:
[1] workflowr_1.7.2
loaded via a namespace (and not attached):
[1] vctrs_0.6.5 httr_1.4.7 cli_3.6.5 knitr_1.50
[5] rlang_1.1.6 xfun_0.53 stringi_1.8.7 processx_3.8.6
[9] promises_1.3.3 jsonlite_2.0.0 glue_1.8.0 rprojroot_2.1.1
[13] git2r_0.36.2 htmltools_0.5.8.1 httpuv_1.6.16 ps_1.9.1
[17] sass_0.4.10 rmarkdown_2.29 jquerylib_0.1.4 tibble_3.3.0
[21] evaluate_1.0.5 fastmap_1.2.0 yaml_2.3.10 lifecycle_1.0.4
[25] whisker_0.4.1 stringr_1.5.2 compiler_4.5.1 fs_1.6.6
[29] pkgconfig_2.0.3 Rcpp_1.1.0 rstudioapi_0.17.1 later_1.4.4
[33] digest_0.6.37 R6_2.6.1 pillar_1.11.0 callr_3.7.6
[37] magrittr_2.0.4 bslib_0.9.0 tools_4.5.1 cachem_1.1.0
[41] getPass_0.2-4