Using crumblr in practice

Here we consider counts for 8 cell types from quantified using single cell RNA-seq data from unstimulated and interferon β stimulated PBMCs from 8 subjects (Kang, et al., 2018).

The functions here incorporate the precision weights:

Installation

To install this package, start R and enter:

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

# Select release #1 or #2

# 1) Bioconductor release (coming soon...)
# BiocManager::install("crumblr")

# 2) Latest stable release
devtools::install_github("DiseaseNeurogenomics/crumblr")

Process data

Here we evaluate whether the observed cell proportions change in response to interferon β. Given the results here, we cannot reject the null hypothesis that interferon β does not affect the cell type proportions.

library(crumblr)

# Load cell counts, clustering and metadata
# from Kang, et al. (2018) https://doi.org/10.1038/nbt.4042
data(IFNCellCounts)

# Apply crumblr transformation
# cobj is an EList object compatable with limma workflow
# cobj$E stores transformed values
# cobj$weights stores precision weights
#    corresponding to the regularized inverse variance
cobj <- crumblr( df_cellCounts )

Variance partitioning

Decomposing the variance illustrates that more variation is explained by subject than stimulation status.

library(variancePartition)

# Partition variance into components for Subject (i.e. ind)
#   and stimulation status, and residual variation
form =  ~ (1|ind) + (1|StimStatus)
vp <- fitExtractVarPartModel(cobj, form, info)

# Plot variance fractions
fig.vp = plotPercentBars(vp)
fig.vp

PCA

Performing PCA on the transformed cell counts indicates that the samples cluster based on subject rather than stimulation status.

library(ggplot2)

# Perform PCA
# use crumblr::standardize() to get values with 
# approximately equal sampling variance,
# which is a key property for downstream PCA and clustering analysis.
pca <- prcomp(t(standardize(cobj)))

# merge with metadata
df_pca <- merge(pca$x, info, by = "row.names")

# Plot PCA
#   color by Subject
#   shape by Stimulated vs unstimulated
ggplot(df_pca, aes(PC1, PC2, color = as.character(ind), shape = StimStatus)) +
  geom_point(size = 3) +
  theme_classic() +
  theme(aspect.ratio = 1) +
  scale_color_discrete(name = "Subject") +
  xlab("PC1") +
  ylab("PC2")

Hierachical clustering

The samples from the same subject also cluster together.

heatmap(cobj$E)

Differential testing

# Use variancePartition workflow to analyze each cell type
# Perform regression on each cell type separately
#  then use eBayes to shrink residual variance
# Also compatible with limma::lmFit()
fit <- dream(cobj, ~ StimStatus + ind, info)
fit <- eBayes(fit)

# Extract results for each cell type
topTable(fit, coef = "StimStatusstim", number = Inf)

##                         logFC    AveExpr          t     P.Value  adj.P.Val         B
## CD8 T cells       -0.25085170  0.0857175 -4.0787416 0.002436375 0.01949100 -1.279815
## Dendritic cells    0.37386979 -2.1849234  3.1619195 0.010692544 0.02738587 -2.638507
## CD14+ Monocytes   -0.10525402  1.2698117 -3.1226341 0.011413912 0.02738587 -2.709377
## B cells           -0.10478652  0.5516882 -3.0134349 0.013692935 0.02738587 -2.940542
## CD4 T cells       -0.07840101  2.0201947 -2.2318104 0.050869691 0.08139151 -4.128069
## FCGR3A+ Monocytes  0.07425165 -0.2567492  1.6647681 0.128337022 0.17111603 -4.935304
## NK cells           0.10270672  0.3797777  1.5181860 0.161321761 0.18436773 -5.247806
## Megakaryocytes     0.01377768 -1.8655172  0.1555131 0.879651456 0.87965146 -6.198336

Multivariate testing along a tree

We can gain power by jointly testing multiple cell types using a multivariate statistical model, instead of testing one cell type at a time. Here we construct a hierarchical clustering between cell types based on gene expression from pseudobulk, and perform a multivariate test for each internal node of the tree based on its leaf nodes. The results for the leaves are the same as from topTable() above. At each internal node treeTest() performs a fixed effects meta-analysis of the coefficients of the leaves while modeling the covariance between coefficient estimates. In the backend, this is implemented using variancePartition::mvTest() and remaCor package.

# Perform multivariate test across the hierarchy
res <- treeTest(fit, cobj, hcl, coef = "StimStatusstim")

# Plot hierarchy and testing results
plotTreeTest(res)

# Plot hierarchy and regression coefficients
plotTreeTestBeta(res)

Combined plotting

plotTreeTestBeta(res) + 
  theme(legend.position="bottom", legend.box = "vertical") | 
  plotForest(res, hide=FALSE) | 
  fig.vp

Developed by Gabriel Hoffman

Run on 2024-06-17 13:35:54.230155