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README.Rmd
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An example how to build multiple super-cell-like objects, including ‘exact’ (Super-cells obtained with the exact coarse-gaining), ‘approx’ (Super-cells obtained with the aaproximate coarse-graining), ’metacell(.*)’ (Metacell build on the same genes as super-cells – ‘metacell_SC_like’; and Metacell build in a default set of genes – ‘metacell_default’) for a set of graining levels and random seeds.

# if (!requireNamespace("BiocManager", quietly = TRUE))
#     install.packages("BiocManager")
# 
# BiocManager::install("SingleCellExperiment")
# 
# if (!requireNamespace("remotes")) install.packages("remotes")
# remotes::install_github("GfellerLab/SuperCell")
# remotes::install_github("mariiabilous/SuperCellBM")

library(SingleCellExperiment)
library(SuperCell)
library(SuperCellBM)

Load some default parameters

Such as .gamma.seqfor the set of fraining levels, .seed.seq for the set of random seeds, adata.folder and fig.folder for the folders where to write data and plots. For the full list of the default parameters, see ./examples/config/Tian_config.R.

source("./examples/config/Tian_config.R")

Flags

Whether to compute super-cell (ToComputeSC) or whether to compute super-cell gene expression (ToComputeSC_GE) or load saved files. Make sure, these file exists :) Flag ToTestPackage is used to run stript in 2 modes: package testing (ToTestPackage == TRUE) or generating super-cell structure and super-cell gene expression for the further analyses (ToTestPackage == FALSE).

ToComputeSC <- T
ToComputeSC_GE <- T

ToTestPackage <- T # @Loc, This is just to test whether package works (on reduced set of graining levels and seeds, turn it to FALSE to get real data @ all grainig levels and seeds)

filename_suf <- "" # variable to add a suffix to the saved files in case of testing of the package

if(ToTestPackage){
  testing_gamma_seq <- c(1, 10, 100)
  testing_seed_seq <- .seed.seq[1:3]
  
  warning(paste("The reduced set of graining leveles and seeds will be used, to get real output, turn it ti FALSE"))
  warning(paste("Original set of graining levels is:", paste(.gamma.seq, collapse = ", "), 
                "but used testing set is:", paste(testing_gamma_seq, collapse = ", ")))
  warning(paste("Original set of seeds  is:", paste(.seed.seq, collapse = ", "), 
                "but used testing set is:", paste(testing_seed_seq, collapse = ", ")))
  
  .gamma.seq <- testing_gamma_seq
  .seed.seq <- testing_seed_seq
  
  filename_suf = "_testing_package"
}
## Warning: The reduced set of graining leveles and seeds will be used, to get real
## output, turn it ti FALSE

## Warning: Original set of graining levels is: 1, 2, 5, 10, 20, 50, 100, 200 but
## used testing set is: 1, 10, 100

## Warning: Original set of seeds is: 12345, 111, 19, 42, 7, 559241, 123, 987, 234,
## 91, 877, 451, 817 but used testing set is: 12345, 111, 19

Load cell_lines data from Tian et al., 2019.

RData.file.path <- file.path(data.folder, 'cell_lines_git.RData')

if(!file.exists(RData.file.path)){
  if(!dir.exists(data.folder)) dir.create(data.folder, recursive = T)
  download.file('https://github.com/LuyiTian/sc_mixology/blob/master/data/sincell_with_class_5cl.RData?raw=true', 
                RData.file.path)
}

load(RData.file.path)

# keep used dataset 
cell_lines_SCE <- sce_sc_10x_5cl_qc

#remove not-used datasets
rm(sc_Celseq2_5cl_p1, sc_Celseq2_5cl_p2, sc_Celseq2_5cl_p3, sce_sc_10x_5cl_qc)

Get and set the main variables, such as single-cell gene expression (sc.GE), single-cell counts (sc.counts), number of single cells (N.c) and total number of genes (N.g). Set matrix column names to cellIDs (cell.ids) and row names to gene names (gene.names).

cell.ids     <- cell_lines_SCE@colData@rownames
N.c          <- cell_lines_SCE@colData@nrows 


gene.names   <- cell_lines_SCE@int_elementMetadata$external_gene_name
N.g          <- length(gene.names)

sc.GE           <- cell_lines_SCE@assays$data$logcounts
colnames(sc.GE) <- cell.ids
rownames(sc.GE) <- gene.names

sc.counts   <- cell_lines_SCE@assays$data$counts
colnames(sc.counts) <- cell.ids
rownames(sc.counts) <- gene.names

## this is not needed at this point, but will be used later
GT.cell.type <- cell_lines_SCE@colData$cell_line_demuxlet
names(GT.cell.type) <- cell.ids
N.clusters         <- length(unique(GT.cell.type))

GT.cell.type.names          <- names(table(GT.cell.type))
GT.cell.type.2.num          <- 1:length(unique(GT.cell.type))
names(GT.cell.type.2.num)   <- GT.cell.type.names
GT.cell.type.num            <- GT.cell.type.2.num[GT.cell.type]
names(GT.cell.type.num)     <- names(GT.cell.type)

## uncomment this when needed 
#.pal.GT <- .color.tsne.Tian  ## to Global Config
#scales::show_col(.pal.GT)


#mito.genes <-  grep(pattern = "^MT", x = gene.names, value = TRUE)
#ribo.genes <-  grep(pattern = "^RP[LS]", x = gene.names, value = TRUE)
#mito.ribo.genes <- c(mito.genes, ribo.genes)
#length(mito.ribo.genes)

#gene.meta <- data.frame(name = gene.names, inNcells = rowSums(sc.GE>0), mean.expr = rowMeans(sc.GE), sd = rowSds(sc.GE))
#head(gene.meta)

Compute Super-cells structure

for the Exact, Aprox (Super-cells obtained with the exact or approximate coarse-graining), Subsampling or Random (random grouping of cells into super-cells).

filename <- paste0('initial', filename_suf)

SC.list <- compute_supercells(
  sc.GE,
  ToComputeSC = ToComputeSC,
  data.folder = data.folder,
  filename = filename,
  gamma.seq = .gamma.seq,
  n.var.genes = .N.var.genes,
  k.knn = .k.knn,
  n.pc = .N.comp,
  approx.N = .approx.N,
  fast.pca = TRUE,
  genes.use = .genes.use, 
  genes.exclude = .genes.omit,
  seed.seq = .seed.seq
  )

cat(paste("Super-cell computed for:", paste(names(SC.list), collapse = ", "), 
          "\nat graining levels:", paste(names(SC.list[['Approx']]), collapse = ", "),
          "\nfor seeds:", paste(names(SC.list[['Approx']][[1]]), collapse = ", "), "\n",
          "\nand saved to / loaded from", paste0(filename, ".Rds")))
## Super-cell computed for: Exact, Approx, Random, Subsampling 
## at graining levels: 1, 10, 100 
## for seeds: 12345, 111, 19 
##  
## and saved to / loaded from initial_testing_package.Rds

Compute metacells in two settings:

  • ‘metacell_default’ - when metacell is computed with the default parameters (from the tutorial), using gene set filtered by MC
  • ‘metacell_SC_like’ - when metacell is computed with the same default parameters, but at the same set of genes as Super-cells
SC.mc <- compute_supercells_metacells(
  sc.counts = sc.counts, 
  gamma.seq = .gamma.seq,
  SC.list = SC.list,
  proj.name = proj.name,
  ToComputeSC = ToComputeSC, 
  mc.k.knn = 100,
  T_vm_def = 0.08,
  MC.folder = "MC", 
  MC_gene_settings = c('metacell_default', 'metacell_SC_like') # do not change
)

Get actual graining levels obtained with Metacell

additional_gamma_seq <- get_actual_gammas_metacell(SC.mc)

cat(paste("Metacells were computed in", length(names(SC.mc)), "settings:", paste(names(SC.mc), collapse = ", "), 
          "\nfor Gammas:", paste(names(SC.mc[[1]]), collapse = ", "), 
          "\nbut actual gammas are:", paste(additional_gamma_seq, collapse = ", ")
))
## Metacells were computed in 2 settings: metacell_default, metacell_SC_like 
## for Gammas: 1, 10, 100 
## but actual gammas are: 46, 54, 69

# manually expand MC because later we will have 2 different setting for MC profile: fp - footpring of MC, av - averaged 
SC.mc.fp <- SC.mc
names(SC.mc.fp) <- sapply(names(SC.mc), FUN = function(x){paste0(x, '_fp')})

SC.mc.av <- SC.mc
names(SC.mc.av) <- sapply(names(SC.mc), FUN = function(x){paste0(x, '_av')})

SC.mc.expanded <- c(SC.mc.fp, SC.mc.av)

names(SC.mc.expanded)
## [1] "metacell_default_fp" "metacell_SC_like_fp" "metacell_default_av"
## [4] "metacell_SC_like_av"

rm(SC.mc.fp, SC.mc.av, SC.mc)

Compute super-cells (Exact, Approx, Subsampling and Random) at the addidional grainig levels obtained with Metacell.

So that we can dirrectly compare the results of Super-cells and Metacells at the same graining levels.

filename <- paste0('additional_gammas', filename_suf)

SC.list <- compute_supercells_additional_gammas(
  SC.list,
  additional_gamma_seq = additional_gamma_seq,
  ToComputeSC = ToComputeSC,
  data.folder = data.folder,
  filename = filename,
  approx.N = .approx.N,
  fast.pca = TRUE
)

cat(paste("Super-cells of methods:", paste(names(SC.list), collapse = ", "), 
      "\nwere computed at aggitional graining levels:", paste(additional_gamma_seq, collapse = ", "), 
      "\nand added to SC.list"
      ))
## Super-cells of methods: Exact, Approx, Random, Subsampling 
## were computed at aggitional graining levels: 46, 54, 69 
## and added to SC.list

Concatenate Metacells to the list of Super-cells

SC.list <- c(SC.list, SC.mc.expanded)
rm(SC.mc.expanded)

filename <- paste0("all", filename_suf)
saveRDS(SC.list, file = file.path(data.folder, "SC", paste0(filename, ".Rds")))

cat(paste(
  "Metacell data added to SC.list \nand now it contains:",
  paste(names(SC.list), collapse = ", "),
  "\nSC.list was saved to", file.path(data.folder, "SC", paste0(filename, ".Rds"))
))
## Metacell data added to SC.list 
## and now it contains: Exact, Approx, Random, Subsampling, metacell_default_fp, metacell_SC_like_fp, metacell_default_av, metacell_SC_like_av 
## SC.list was saved to examples/data/Tian/SC/all_testing_package.Rds

Compute GE for Super-cell data

GE profile for the super-cell data is computede:

  • for super-cells (Exact, Approx) by averaging gene expression within super-cells,
  • for Random, also averaging gene expression within super-cells
  • for the Subsampling, sc.GE matrix is just subsampled,
  • for Metacells, gene expression is computed in 2 ways:
    1. the same as super-cells (averaging gene expression within Metacells) -> metacell_(.*)_av
    2. using the default output of Metacell maned footprint -> metacell_(.*)_fp
filename <- paste("all", filename_suf)

SC.GE.list <- compute_supercells_GE(
  sc.GE = sc.GE, 
  SC.list = SC.list,
  ToComputeSC_GE = ToComputeSC_GE, 
  data.folder = data.folder,
  filename = filename
)

cat(paste("Gene expression profile computed for:", paste(names(SC.GE.list), collapse = ", "), 
    "\nat graining levels:", paste(sort(as.numeric(names(SC.GE.list[['Approx']]))), collapse = ", "),
    "\nfor seeds:", paste(names(SC.GE.list[['Approx']][[1]]), collapse = ", "),
    "\nand saved to / loaded from", paste0(filename, ".Rds")
    ))
## Gene expression profile computed for: Exact, Approx, Random, Subsampling, metacell_default_fp, metacell_SC_like_fp, metacell_default_av, metacell_SC_like_av 
## at graining levels: 1, 10, 46, 54, 69, 100 
## for seeds: 12345, 111, 19 
## and saved to / loaded from all _testing_package.Rds

Final

## [1] "Done! Congrats!"

## Warning: (!) Script was run in a test mode, to get real cell_line super-cell
## data, run this script with ToTestPackage <- FALSE

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