1 Introduction

Since read counts are summed across cells in a pseudobulk approach, modeling continuous cell-level covariates also requires a collapsing step. Here we summarize the values of a variable from a set of cells using the mean, and store the value for each cell type. Including these variables in a regression formula uses the summarized values from the corresponding cell type.

We demonstrate this feature on a lightly modified analysis of PBMCs from 8 individuals stimulated with interferon-β (Kang, et al, 2018, Nature Biotech).

2 Standard processing

Here is the code from the main vignette:

library(dreamlet)
library(muscat)
library(ExperimentHub)
library(scater)

# Download data, specifying EH2259 for the Kang, et al study
eh <- ExperimentHub()
sce <- eh[["EH2259"]]

# only keep singlet cells with sufficient reads
sce <- sce[rowSums(counts(sce) > 0) > 0, ]
sce <- sce[, colData(sce)$multiplets == "singlet"]

# compute QC metrics
qc <- perCellQCMetrics(sce)

# remove cells with few or many detected genes
ol <- isOutlier(metric = qc$detected, nmads = 2, log = TRUE)
sce <- sce[, !ol]

# set variable indicating stimulated (stim) or control (ctrl)
sce$StimStatus <- sce$stim

In many datasets, continuous cell-level variables could be mapped reads, gene count, mitochondrial rate, etc. There are no continuous cell-level variables in this dataset, so we can simulate two from a normal distribution:

sce$value1 <- rnorm(ncol(sce))
sce$value2 <- rnorm(ncol(sce))

3 Pseudobulk

Now compute the pseudobulk using standard code:

sce$id <- paste0(sce$StimStatus, sce$ind)

# Create pseudobulk
pb <- aggregateToPseudoBulk(sce,
  assay = "counts",
  cluster_id = "cell",
  sample_id = "id",
  verbose = FALSE
)

The means per variable, cell type, and sample are stored in the pseudobulk SingleCellExperiment object:

metadata(pb)$aggr_means
## # A tibble: 128 × 5
## # Groups:   cell [8]
##    cell    id       cluster  value1   value2
##    <fct>   <fct>      <dbl>   <dbl>    <dbl>
##  1 B cells ctrl101     3.96  0.0351 -0.0174 
##  2 B cells ctrl1015    4.00  0.0214  0.0133 
##  3 B cells ctrl1016    4    -0.0665  0.0260 
##  4 B cells ctrl1039    4.04 -0.0933  0.195  
##  5 B cells ctrl107     4    -0.0147 -0.0703 
##  6 B cells ctrl1244    4    -0.0586  0.0222 
##  7 B cells ctrl1256    4.01 -0.0172 -0.0591 
##  8 B cells ctrl1488    4.02 -0.188   0.0879 
##  9 B cells stim101     4.09 -0.0725 -0.0534 
## 10 B cells stim1015    4.06 -0.0770 -0.00699
## # ℹ 118 more rows

4 Analysis

Including these variables in a regression formula uses the summarized values from the corresponding cell type. This happens behind the scenes, so the user doesn’t need to distinguish bewteen sample-level variables stored in colData(pb) and cell-level variables stored in metadata(pb)$aggr_means.

Variance partition and hypothesis testing proceeds as ususal:

form <- ~ StimStatus + value1 + value2

# Normalize and apply voom/voomWithDreamWeights
res.proc <- processAssays(pb, form, min.count = 5)

# run variance partitioning analysis
vp.lst <- fitVarPart(res.proc, form)

# Summarize variance fractions genome-wide for each cell type
plotVarPart(vp.lst, label.angle = 60)

# Differential expression analysis within each assay
res.dl <- dreamlet(res.proc, form)

# dreamlet results include coefficients for value1 and value2
res.dl
## class: dreamletResult 
## assays(8): B cells CD14+ Monocytes ... Megakaryocytes NK cells
## Genes:
##  min: 164 
##  max: 5262 
## details(7): assay n_retain ... n_errors error_initial
## coefNames(4): (Intercept) StimStatusstim value1 value2

5 Session Info

## R version 4.4.1 (2024-06-14)
## Platform: aarch64-apple-darwin20
## Running under: macOS Ventura 13.6.7
## 
## Matrix products: default
## BLAS:   /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib 
## LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib;  LAPACK version 3.12.0
## 
## locale:
## [1] C/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
## 
## time zone: America/New_York
## tzcode source: internal
## 
## attached base packages:
## [1] stats4    stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
##  [1] muscData_1.19.0             scater_1.34.0              
##  [3] scuttle_1.16.0              ExperimentHub_2.14.0       
##  [5] AnnotationHub_3.14.0        BiocFileCache_2.14.0       
##  [7] dbplyr_2.5.0                muscat_1.20.0              
##  [9] dreamlet_1.4.1              SingleCellExperiment_1.28.0
## [11] SummarizedExperiment_1.36.0 Biobase_2.66.0             
## [13] GenomicRanges_1.58.0        GenomeInfoDb_1.42.0        
## [15] IRanges_2.40.0              S4Vectors_0.44.0           
## [17] BiocGenerics_0.52.0         MatrixGenerics_1.18.0      
## [19] matrixStats_1.4.1           variancePartition_1.36.2   
## [21] BiocParallel_1.40.0         limma_3.62.1               
## [23] ggplot2_3.5.1               BiocStyle_2.34.0           
## 
## loaded via a namespace (and not attached):
##   [1] bitops_1.0-9              httr_1.4.7               
##   [3] RColorBrewer_1.1-3        doParallel_1.0.17        
##   [5] Rgraphviz_2.50.0          numDeriv_2016.8-1.1      
##   [7] sctransform_0.4.1         tools_4.4.1              
##   [9] backports_1.5.0           utf8_1.2.4               
##  [11] R6_2.5.1                  metafor_4.6-0            
##  [13] mgcv_1.9-1                GetoptLong_1.0.5         
##  [15] withr_3.0.2               prettyunits_1.2.0        
##  [17] gridExtra_2.3             cli_3.6.3                
##  [19] sandwich_3.1-1            labeling_0.4.3           
##  [21] sass_0.4.9                KEGGgraph_1.66.0         
##  [23] SQUAREM_2021.1            mvtnorm_1.3-2            
##  [25] blme_1.0-6                mixsqp_0.3-54            
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##  [33] gtools_3.9.5              dplyr_1.1.4              
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## [119] RhpcBLASctl_0.23-42       htmltools_0.5.8.1        
## [121] pkgconfig_2.0.3           lme4_1.1-35.5            
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## [131] jquerylib_0.1.4           zoo_1.8-12               
## [133] jsonlite_1.8.9            BiocSingular_1.22.0      
## [135] RCurl_1.98-1.16           magrittr_2.0.3           
## [137] GenomeInfoDbData_1.2.13   munsell_0.5.1            
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## [147] plyr_1.8.9                listenv_0.9.1            
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## [173] xtable_1.8-4              fANCOVA_0.6-1            
## [175] viridisLite_0.4.2         truncnorm_1.0-9          
## [177] tibble_3.2.1              lmerTest_3.1-3           
## [179] glmmTMB_1.1.10            memoise_2.0.1            
## [181] beeswarm_0.4.0            AnnotationDbi_1.68.0     
## [183] cluster_2.1.6             globals_0.16.3           
## [185] GSEABase_1.68.0