Getting started with SimBu

Alexander Dietrich

Installation

To install the developmental version of the package, run:

install.packages("devtools")
devtools::install_github("omnideconv/SimBu")

To install from Bioconductor:

if (!require("BiocManager", quietly = TRUE)) {
  install.packages("BiocManager")
}

BiocManager::install("SimBu")
library(SimBu)

Introduction

As complex tissues are typically composed of various cell types, deconvolution tools have been developed to computationally infer their cellular composition from bulk RNA sequencing (RNA-seq) data. To comprehensively assess deconvolution performance, gold-standard datasets are indispensable. Gold-standard, experimental techniques like flow cytometry or immunohistochemistry are resource-intensive and cannot be systematically applied to the numerous cell types and tissues profiled with high-throughput transcriptomics. The simulation of ‘pseudo-bulk’ data, generated by aggregating single-cell RNA-seq (scRNA-seq) expression profiles in pre-defined proportions, offers a scalable and cost-effective alternative. This makes it feasible to create in silico gold standards that allow fine-grained control of cell-type fractions not conceivable in an experimental setup. However, at present, no simulation software for generating pseudo-bulk RNA-seq data exists.
SimBu was developed to simulate pseudo-bulk samples based on various simulation scenarios, designed to test specific features of deconvolution methods. A unique feature of SimBu is the modelling of cell-type-specific mRNA bias using experimentally-derived or data-driven scaling factors. Here, we show that SimBu can generate realistic pseudo-bulk data, recapitulating the biological and statistical features of real RNA-seq data. Finally, we illustrate the impact of mRNA bias on the evaluation of deconvolution tools and provide recommendations for the selection of suitable methods for estimating mRNA content.

Getting started

This chapter covers all you need to know to quickly simulate some pseudo-bulk samples!

This package can simulate samples from local or public data. This vignette will work with artificially generated data as it serves as an overview for the features implemented in SimBu. For the public data integration using sfaira (Fischer et al. 2020), please refer to the “Public Data Integration” vignette.

We will create some toy data to use for our simulations; two matrices with 300 cells each and 1000 genes/features. One represents raw count data, while the other matrix represents scaled TPM-like data. We will assign these cells to some immune cell types.

counts <- Matrix::Matrix(matrix(stats::rpois(3e5, 5), ncol = 300), sparse = TRUE)
tpm <- Matrix::Matrix(matrix(stats::rpois(3e5, 5), ncol = 300), sparse = TRUE)
tpm <- Matrix::t(1e6 * Matrix::t(tpm) / Matrix::colSums(tpm))
colnames(counts) <- paste0("cell_", rep(1:300))
colnames(tpm) <- paste0("cell_", rep(1:300))
rownames(counts) <- paste0("gene_", rep(1:1000))
rownames(tpm) <- paste0("gene_", rep(1:1000))
annotation <- data.frame(
  "ID" = paste0("cell_", rep(1:300)),
  "cell_type" = c(
    rep("T cells CD4", 50),
    rep("T cells CD8", 50),
    rep("Macrophages", 100),
    rep("NK cells", 10),
    rep("B cells", 70),
    rep("Monocytes", 20)
  )
)

Creating a dataset

SimBu uses the SummarizedExperiment class as storage for count data as well as annotation data. Currently it is possible to store two matrices at the same time: raw counts and TPM-like data (this can also be some other scaled count matrix, such as RPKM, but we recommend to use TPMs). These two matrices have to have the same dimensions and have to contain the same genes and cells. Providing the raw count data is mandatory!
SimBu scales the matrix that is added via the tpm_matrix slot by default to 1e6 per cell, if you do not want this, you can switch it off by setting the scale_tpm parameter to FALSE. Additionally, the cell type annotation of the cells has to be given in a dataframe, which has to include the two columns ID and cell_type. If additional columns from this annotation should be transferred to the dataset, simply give the names of them in the additional_cols parameter.

To generate a dataset that can be used in SimBu, you can use the dataset() method; other methods exist as well, which are covered in the “Inputs & Outputs” vignette.

ds <- SimBu::dataset(
  annotation = annotation,
  count_matrix = counts,
  tpm_matrix = tpm,
  name = "test_dataset"
)
#> Filtering genes...
#> Created dataset.

SimBu offers basic filtering options for your dataset, which you can apply during dataset generation:

Simulate pseudo bulk datasets

We are now ready to simulate the first pseudo bulk samples with the created dataset:

simulation <- SimBu::simulate_bulk(
  data = ds,
  scenario = "random",
  scaling_factor = "NONE",
  ncells = 100,
  nsamples = 10,
  BPPARAM = BiocParallel::MulticoreParam(workers = 4), # this will use 4 threads to run the simulation
  run_parallel = TRUE
) # multi-threading to TRUE
#> Using parallel generation of simulations.
#> Warning in BiocParallel::MulticoreParam(workers = 4): MulticoreParam() not
#> supported on Windows, use SnowParam()
#> Finished simulation.

ncells sets the number of cells in each sample, while nsamples sets the total amount of simulated samples.
If you want to simulate a specific sequencing depth in your simulations, you can use the total_read_counts parameter to do so. Note that this parameter is only applied on the counts matrix (if supplied), as TPMs will be scaled to 1e6 by default.

SimBu can add mRNA bias by using different scaling factors to the simulations using the scaling_factor parameter. A detailed explanation can be found in the “Scaling factor” vignette.

Currently there are 6 scenarios implemented in the package:

pure_scenario_dataframe <- data.frame(
  "B cells" = c(0.2, 0.1, 0.5, 0.3),
  "T cells" = c(0.3, 0.8, 0.2, 0.5),
  "NK cells" = c(0.5, 0.1, 0.3, 0.2),
  row.names = c("sample1", "sample2", "sample3", "sample4")
)
pure_scenario_dataframe
#>         B.cells T.cells NK.cells
#> sample1     0.2     0.3      0.5
#> sample2     0.1     0.8      0.1
#> sample3     0.5     0.2      0.3
#> sample4     0.3     0.5      0.2

Results

The simulation object contains three named entries:

utils::head(SummarizedExperiment::assays(simulation$bulk)[["bulk_counts"]])
#> 6 x 10 sparse Matrix of class "dgCMatrix"
#>   [[ suppressing 10 column names 'random_sample1', 'random_sample2', 'random_sample3' ... ]]
#>                                               
#> gene_1 513 550 557 488 540 583 518 558 514 485
#> gene_2 503 457 506 515 490 508 464 465 520 477
#> gene_3 485 480 519 483 472 497 511 513 516 520
#> gene_4 532 516 575 512 482 538 507 488 566 500
#> gene_5 546 480 498 479 493 541 532 511 544 539
#> gene_6 528 491 534 536 460 531 598 511 495 498
utils::head(SummarizedExperiment::assays(simulation$bulk)[["bulk_tpm"]])
#> 6 x 10 sparse Matrix of class "dgCMatrix"
#>   [[ suppressing 10 column names 'random_sample1', 'random_sample2', 'random_sample3' ... ]]
#>                                                                             
#> gene_1 1016.9016 1007.5008  993.7881 1092.2194 1051.1695  994.9660 1022.6753
#> gene_2  905.4864  943.4580  938.6533 1035.2110  956.9343  929.7308  996.4019
#> gene_3  884.7450 1001.5147  864.4843  925.8676  924.6009  921.7638  949.8533
#> gene_4  880.5735 1115.9696  983.6763  828.3940  995.4841 1078.6742 1048.1623
#> gene_5 1048.8868 1086.9301  964.9968  986.9180 1042.3529 1084.6112  981.8453
#> gene_6  955.2776  987.5368 1050.2272 1091.2313 1011.8135 1037.3142 1049.3152
#>                                     
#> gene_1 1051.6245 1024.0751 1051.7815
#> gene_2  960.0764 1026.0266  941.1532
#> gene_3 1023.2663  930.6924  971.4474
#> gene_4  981.5937 1056.5400  972.3125
#> gene_5  960.1552 1073.4474 1070.1151
#> gene_6  964.5299 1006.2980 1029.1135

If only a single matrix was given to the dataset initially, only one assay is filled.

It is also possible to merge simulations:

simulation2 <- SimBu::simulate_bulk(
  data = ds,
  scenario = "even",
  scaling_factor = "NONE",
  ncells = 1000,
  nsamples = 10,
  BPPARAM = BiocParallel::MulticoreParam(workers = 4),
  run_parallel = TRUE
)
#> Using parallel generation of simulations.
#> Warning in BiocParallel::MulticoreParam(workers = 4): MulticoreParam() not
#> supported on Windows, use SnowParam()
#> Finished simulation.
merged_simulations <- SimBu::merge_simulations(list(simulation, simulation2))

Finally here is a barplot of the resulting simulation:

SimBu::plot_simulation(simulation = merged_simulations)

More features

Simulate using a whitelist (and blacklist) of cell-types

Sometimes, you are only interested in specific cell-types (for example T cells), but the dataset you are using has too many other cell-types; you can handle this issue during simulation using the whitelist parameter:

simulation <- SimBu::simulate_bulk(
  data = ds,
  scenario = "random",
  scaling_factor = "NONE",
  ncells = 1000,
  nsamples = 20,
  BPPARAM = BiocParallel::MulticoreParam(workers = 4),
  run_parallel = TRUE,
  whitelist = c("T cells CD4", "T cells CD8")
)
#> Using parallel generation of simulations.
#> Warning in BiocParallel::MulticoreParam(workers = 4): MulticoreParam() not
#> supported on Windows, use SnowParam()
#> Finished simulation.
SimBu::plot_simulation(simulation = simulation)

In the same way, you can also provide a blacklist parameter, where you name the cell-types you don’t want to be included in your simulation.

utils::sessionInfo()
#> R version 4.4.1 (2024-06-14 ucrt)
#> Platform: x86_64-w64-mingw32/x64
#> Running under: Windows Server 2022 x64 (build 20348)
#> 
#> Matrix products: default
#> 
#> 
#> locale:
#> [1] LC_COLLATE=C                          
#> [2] LC_CTYPE=English_United States.utf8   
#> [3] LC_MONETARY=English_United States.utf8
#> [4] LC_NUMERIC=C                          
#> [5] LC_TIME=English_United States.utf8    
#> 
#> time zone: America/New_York
#> tzcode source: internal
#> 
#> attached base packages:
#> [1] stats     graphics  grDevices utils     datasets  methods   base     
#> 
#> other attached packages:
#> [1] SimBu_1.8.0
#> 
#> loaded via a namespace (and not attached):
#>  [1] SummarizedExperiment_1.36.0 gtable_0.3.6               
#>  [3] xfun_0.48                   bslib_0.8.0                
#>  [5] ggplot2_3.5.1               Biobase_2.66.0             
#>  [7] lattice_0.22-6              vctrs_0.6.5                
#>  [9] tools_4.4.1                 generics_0.1.3             
#> [11] stats4_4.4.1                parallel_4.4.1             
#> [13] tibble_3.2.1                fansi_1.0.6                
#> [15] highr_0.11                  pkgconfig_2.0.3            
#> [17] Matrix_1.7-1                data.table_1.16.2          
#> [19] RColorBrewer_1.1-3          S4Vectors_0.44.0           
#> [21] sparseMatrixStats_1.18.0    lifecycle_1.0.4            
#> [23] GenomeInfoDbData_1.2.13     compiler_4.4.1             
#> [25] farver_2.1.2                munsell_0.5.1              
#> [27] codetools_0.2-20            GenomeInfoDb_1.42.0        
#> [29] htmltools_0.5.8.1           sass_0.4.9                 
#> [31] yaml_2.3.10                 pillar_1.9.0               
#> [33] crayon_1.5.3                jquerylib_0.1.4            
#> [35] tidyr_1.3.1                 BiocParallel_1.40.0        
#> [37] DelayedArray_0.32.0         cachem_1.1.0               
#> [39] abind_1.4-8                 tidyselect_1.2.1           
#> [41] digest_0.6.37               dplyr_1.1.4                
#> [43] purrr_1.0.2                 labeling_0.4.3             
#> [45] fastmap_1.2.0               grid_4.4.1                 
#> [47] colorspace_2.1-1            cli_3.6.3                  
#> [49] SparseArray_1.6.0           magrittr_2.0.3             
#> [51] S4Arrays_1.6.0              utf8_1.2.4                 
#> [53] withr_3.0.2                 UCSC.utils_1.2.0           
#> [55] scales_1.3.0                rmarkdown_2.28             
#> [57] XVector_0.46.0              httr_1.4.7                 
#> [59] matrixStats_1.4.1           proxyC_0.4.1               
#> [61] evaluate_1.0.1              knitr_1.48                 
#> [63] GenomicRanges_1.58.0        IRanges_2.40.0             
#> [65] rlang_1.1.4                 Rcpp_1.0.13                
#> [67] glue_1.8.0                  BiocGenerics_0.52.0        
#> [69] jsonlite_1.8.9              R6_2.5.1                   
#> [71] MatrixGenerics_1.18.0       zlibbioc_1.52.0

References

Fischer, David S., Leander Dony, Martin König, Abdul Moeed, Luke Zappia, Sophie Tritschler, Olle Holmberg, Hananeh Aliee, and Fabian J. Theis. 2020. “Sfaira Accelerates Data and Model Reuse in Single Cell Genomics.” bioRxiv. https://doi.org/10.1101/2020.12.16.419036.