Last updated: 2025-01-09

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The aim of this analysis is to take an initial look at the mouse pancreas endocrinogenesis data from Bastidas-Ponce et al 2019 (see also this GitHub repository) and that was later analyzed in the scVelo paper.

To run the code, you will need to first download the “GSE132188_adata.h5ad.h5” file from the GEO website, accession GSE132188.

Then run the Python script prepare_pancreas_endocrine_data.py to generate the file “pancreas_endocrine_alldays.h5ad”.

First, load the packages needed for this analysis.

library(Matrix)
library(anndata)
library(reticulate)
library(tools)
library(ggplot2)
library(cowplot)
Sys.getenv("RETICULATE_PYTHON")
# [1] "/Users/pcarbo/miniforge3/bin/python"

Note: The AnnData Python package is needed to run this code. I installed anndata 0.11.2 using conda.

Get the count data that was prepared using the Python script.

dat1   <- read_h5ad("../data/pancreas_endocrine_alldays.h5ad")
counts <- dat1$X
counts <- as(counts,"CsparseMatrix")

Get the meta-data downloaded from GEO.

dat2 <- read_h5ad("../data/GSE132188_adata.h5ad.h5")

Align the two data sets.

ids1   <- rownames(dat1$obs)
ids2   <- rownames(dat2$obs)
ids2   <- paste0("e",10*as.numeric(as.character(dat2$obs$day)),"-",ids2)
ids2   <- substr(ids2,1,23)
rows   <- which(is.element(ids1,ids2))
ids1   <- ids1[rows]
counts <- counts[rows,]
obs1   <- dat1$obs[rows,]

Check that the sample ids and genes are the same.

print(all(ids1 == ids2))
print(all(rownames(dat1$var) == rownames(dat2$var)))
# [1] TRUE
# [1] TRUE

Extract the gene info.

gene_info <- dat2$var
gene_info <- cbind(gene = rownames(gene_info),gene_info)
rownames(gene_info) <- NULL

Extract the sample info.

sample_info <- dat2$obs
umap <- dat2$obsm$X_umap
colnames(umap) <- c("umap1","umap2")
sample_info <- cbind(data.frame(id = ids1,stringsAsFactors = FALSE),
                     umap,
                     sample_info)
rownames(sample_info) <- NULL

Save the data and t-SNE results to an .Rdata file for more convenient analysis in R:

save(list = c("gene_info","sample_info","counts"),
     file = "pancreas_endocrine.RData")
resaveRdaFiles("pancreas_endocrine.RData")

The meta-data includes a previously computed UMAP which we can use to visualize of the key structure in the data.

The Bastidas-Ponce et al paper identified 8 main cell types:

cluster_colors <- c("#e41a1c","#377eb8","#4daf4a","#984ea3","#ff7f00",
                    "#ffff33","#a65628","#f781bf")
ggplot(sample_info,
       aes(x = umap1,y = umap2,color = clusters_fig2_final)) +
  geom_point(size = 0.75) +
  scale_color_manual(values = cluster_colors) +
  labs(color = "cluster") +
  theme_cowplot(font_size = 10)

Additionally, they distinguished proliferating vs. non-proliferating cells:

ggplot(sample_info,
       aes(x = umap1,y = umap2,color = proliferation)) +
  geom_point(size = 0.75) +
  scale_color_manual(values = c("dodgerblue","darkblue")) +
  labs(color = "") +
  theme_cowplot(font_size = 10)

Beyond the main cell types and proliferating/non-profilerating, there appears to be additional structure in the data corresponding to the different lineages (days):

lineage_colors <- c("#d01c8b","#f1b6da","#b8e186","#4dac26")
ggplot(sample_info,
       aes(x = umap1,y = umap2,color = day)) +
  geom_point(size = 0.75) +
  scale_color_manual(values = lineage_colors) +
  theme_cowplot(font_size = 10)


sessionInfo()
# R version 4.3.3 (2024-02-29)
# Platform: aarch64-apple-darwin20 (64-bit)
# Running under: macOS Sonoma 14.7.1
# 
# Matrix products: default
# BLAS:   /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRblas.0.dylib 
# LAPACK: /Library/Frameworks/R.framework/Versions/4.3-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.11.0
# 
# 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: America/Chicago
# tzcode source: internal
# 
# attached base packages:
# [1] tools     stats     graphics  grDevices utils     datasets  methods  
# [8] base     
# 
# other attached packages:
# [1] cowplot_1.1.3     ggplot2_3.5.0     reticulate_1.40.0 anndata_0.7.5.6  
# [5] Matrix_1.6-5     
# 
# loaded via a namespace (and not attached):
#  [1] sass_0.4.8       utf8_1.2.4       generics_0.1.3   stringi_1.8.3   
#  [5] lattice_0.22-5   digest_0.6.34    magrittr_2.0.3   evaluate_0.23   
#  [9] grid_4.3.3       fastmap_1.1.1    rprojroot_2.0.4  workflowr_1.7.1 
# [13] jsonlite_1.8.8   whisker_0.4.1    promises_1.2.1   fansi_1.0.6     
# [17] scales_1.3.0     jquerylib_0.1.4  cli_3.6.2        rlang_1.1.3     
# [21] munsell_0.5.0    withr_3.0.0      cachem_1.0.8     yaml_2.3.8      
# [25] dplyr_1.1.4      colorspace_2.1-0 httpuv_1.6.14    assertthat_0.2.1
# [29] vctrs_0.6.5      R6_2.5.1         png_0.1-8        lifecycle_1.0.4 
# [33] git2r_0.33.0     stringr_1.5.1    fs_1.6.3         pkgconfig_2.0.3 
# [37] pillar_1.9.0     bslib_0.6.1      later_1.3.2      gtable_0.3.4    
# [41] glue_1.7.0       Rcpp_1.0.12      highr_0.10       xfun_0.42       
# [45] tibble_3.2.1     tidyselect_1.2.1 knitr_1.45       farver_2.1.1    
# [49] htmltools_0.5.7  rmarkdown_2.26   labeling_0.4.3   compiler_4.3.3