---
title: "Use of SpatialDecon in a small GeoMx dataset"
output:
rmarkdown::html_vignette:
toc: true
vignette: >
%\VignetteIndexEntry{Use of SpatialDecon in a small GeoMx dataet}
%\VignetteEngine{knitr::rmarkdown}
%\VignetteEncoding{UTF-8}
---
<style>
p.caption {
font-size: 1.5em;
}
</style>
```{r, include = FALSE}
knitr::opts_chunk$set(
collapse = TRUE,
comment = "#>"
)
```
### Installation
```{r installation, eval=FALSE}
if(!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
BiocManager::install("SpatialDecon")
```
### Overview
This vignette demonstrates the use of the SpatialDecon package to estimate cell
abundance in spatial gene expression studies.
We'll analyze a small GeoMx dataset from a lung tumor, looking for abundance of
different immune cell types.
This dataset has 30 ROIs. In each ROI, Tumor and Microenvironment segments have
been profiled separately.
### Data preparation
First, we load the package:
```{r setup}
library(SpatialDecon)
```
Now let's load our example data and examine it:
```{r loaddata}
data("mini_geomx_dataset")
norm = mini_geomx_dataset$normalized
raw = mini_geomx_dataset$raw
annot = mini_geomx_dataset$annot
dim(raw)
head(annot)
raw[seq_len(5), seq_len(5)]
# better segment names:
colnames(norm) = colnames(raw) = rownames(annot) =
paste0(annot$ROI, annot$AOI.name)
```
The spatialdecon function takes 3 arguments of expression data:
1. The normalized data.
2. A matrix of expected background for all data points in the normalized
data matrix.
3. Optionally, either a matrix of per-data-point weights, or the raw data,
which is used to derive weights (low counts are less statistically stable,
and this allows spatialdecon to down-weight them.)
We estimate each data point's expected background from the negative control
probes from its corresponding observation:
```{r estimateBG}
# use the NegProbe to estimate per-observation background
per.observation.mean.neg = norm["NegProbe", ]
# and define a background matrix in which each column (observation) is the
# appropriate value of per-observation background:
bg = sweep(norm * 0, 2, per.observation.mean.neg, "+")
dim(bg)
```
A note for background estimation: in studies with two probesets, the genes from
each probeset will have distinct background values, and the above code should be
run separately for each probeset using its corresponding NegProbe value.
Alternatively, the "derive_GeoMx_background" can do this automatically:
```{r derivebg}
bg2 = derive_GeoMx_background(norm = norm,
probepool = rep(1, nrow(norm)),
negnames = "NegProbe")
```
### Cell profile matrices
A "cell profile matrix" is a pre-defined matrix that specifies the expected
expression profiles of each cell type in the experiment.
The SpatialDecon library comes with one such matrix pre-loaded, the "SafeTME"
matrix, designed for estimation of immune and stroma cells in the tumor
microenvironment.
(This matrix was designed to avoid genes commonly expressed by cancer cells;
see the SpatialDecon manuscript for details.)
Let's take a glance at the safeTME matrix:
```{r showsafetme, fig.height=5, fig.width=10, fig.cap = "The safeTME cell profile matrix"}
data("safeTME")
data("safeTME.matches")
signif(safeTME[seq_len(3), seq_len(3)], 2)
heatmap(sweep(safeTME, 1, apply(safeTME, 1, max), "/"),
labRow = NA, margins = c(10, 5))
```
For studies of other tissue types, we have provided a library of cell profile
matrices, available on Github and downloadable with the "download_profile_matrix" function.
For a complete list of matrices, see [CellProfileLibrary GitHub Page](https://github.com/Nanostring-Biostats/CellProfileLibrary)
Below we download a matrix of cell profiles derived from scRNA-seq of a mouse
spleen.
```{r downloadmatrix, fig.height=7, fig.width=10, fig.cap = "The Mouse Spleen profile matrix", eval=T}
mousespleen <- download_profile_matrix(species = "Mouse",
age_group = "Adult",
matrixname = "Spleen_MCA")
dim(mousespleen)
mousespleen[1:4,1:4]
head(cellGroups)
metadata
heatmap(sweep(mousespleen, 1, apply(mousespleen, 1, max), "/"),
labRow = NA, margins = c(10, 5), cexCol = 0.7)
```
For studies where the provided cell profile matrices aren't sufficient or if a
specific single cell dataset is wanted, we can make a custom profile matrix using
the function create_profile_matrix().
This mini single cell dataset is a fraction of the data from Kinchen, J. et al.
Structural Remodeling of the Human Colonic Mesenchyme in Inflammatory Bowel
Disease. Cell 175, 372-386.e17 (2018).
```{r single cell data}
data("mini_singleCell_dataset")
mini_singleCell_dataset$mtx@Dim # genes x cells
as.matrix(mini_singleCell_dataset$mtx)[1:4,1:4]
head(mini_singleCell_dataset$annots)
table(mini_singleCell_dataset$annots$LabeledCellType)
```
**Pericyte cell** and **smooth muscle cell of colon** will be dropped from this
matrix due to low cell count. The average expression across all cells of one type
is returned so the more cells of one type, the better reflection of the true gene
expression. The confidence in these averages can be changed using the minCellNum filter.
```{r creatematrix, fig.height=7, fig.width=10, fig.cap = "Custom profile matrix"}
custom_mtx <- create_profile_matrix(mtx = mini_singleCell_dataset$mtx, # cell x gene count matrix
cellAnnots = mini_singleCell_dataset$annots, # cell annotations with cell type and cell name as columns
cellTypeCol = "LabeledCellType", # column containing cell type
cellNameCol = "CellID", # column containing cell ID/name
matrixName = "custom_mini_colon", # name of final profile matrix
outDir = NULL, # path to desired output directory, set to NULL if matrix should not be written
normalize = FALSE, # Should data be normalized?
minCellNum = 5, # minimum number of cells of one type needed to create profile, exclusive
minGenes = 10, # minimum number of genes expressed in a cell, exclusive
scalingFactor = 5, # what should all values be multiplied by for final matrix
discardCellTypes = TRUE) # should cell types be filtered for types like mitotic, doublet, low quality, unknown, etc.
head(custom_mtx)
heatmap(sweep(custom_mtx, 1, apply(custom_mtx, 1, max), "/"),
labRow = NA, margins = c(10, 5), cexCol = 0.7)
```
Custom matrices can be created from all single cell data classes as long as a
counts matrix and cell annotations can be passed to the function. Here is an
example of creating a matrix using a Seurat object.
```{r createSeuratmatrix}
library(SeuratObject)
library(Seurat)
data("mini_singleCell_dataset")
rownames(mini_singleCell_dataset$annots) <- mini_singleCell_dataset$annots$CellID
seuratObject <- CreateSeuratObject(counts = mini_singleCell_dataset$mtx,
meta.data = mini_singleCell_dataset$annots)
Idents(seuratObject) <- seuratObject$LabeledCellType
rm(mini_singleCell_dataset)
annots <- data.frame(cbind(cellType=as.character(Idents(seuratObject)),
cellID=names(Idents(seuratObject))))
custom_mtx_seurat <- create_profile_matrix(mtx = Seurat::GetAssayData(object = seuratObject,
assay = "RNA",
slot = "counts"),
cellAnnots = annots,
cellTypeCol = "cellType",
cellNameCol = "cellID",
matrixName = "custom_mini_colon",
outDir = NULL,
normalize = FALSE,
minCellNum = 5,
minGenes = 10)
head(custom_mtx_seurat)
paste("custom_mtx and custom_mtx_seurat are identical", all(custom_mtx == custom_mtx_seurat))
```
### Performing basic deconvolution with the spatialdecon function
Now our data is ready for deconvolution.
First we'll show how to use spatialdecon under the basic settings, omitting
optional bells and whistles.
```{r runiss}
res = spatialdecon(norm = norm,
bg = bg,
X = safeTME,
align_genes = TRUE)
str(res)
```
We're most interested in "beta", the matrix of estimated cell abundances.
```{r plotissres, fig.height = 5, fig.width = 8, fig.cap = "Cell abundance estimates"}
heatmap(res$beta, cexCol = 0.5, cexRow = 0.7, margins = c(10,7))
```
### Using the advanced settings of spatialdecon
spatialdecon has several abilities beyond basic deconvolution:
1. If given the nuclei counts for each region/observation, it returns results on
the scale of total cell counts.
2. If given the identities of pure tumor regions/observations, it infers a
handful of tumor-specific expression profiles and appends them to the cell
profile matrix. Doing this accounts for cancer cell-derived expression from any
genes in the cell profile matrix, removing contaminating signal from cancer
cells.
3. If given raw count data, it derives per-data-point weights, using an error
model derived for GeoMx data.
4. If given a "cellmatches" argument, it sums multiple closely-related cell
types into a single score. E.g. if the safeTME matrix is used with the
cell-matching data object "safeTME.matches", it e.g. sums the "T.CD8.naive" and
"T.CD8.memory" scores into a single "CD8.T.cells" score.
Let's take a look at an example cell matching object:
```{r showmatches}
str(safeTME.matches)
```
Now let's run spatialdecon:
```{r runisstils}
# vector identifying pure tumor segments:
annot$istumor = (annot$AOI.name == "Tumor")
# run spatialdecon with all the bells and whistles:
restils = spatialdecon(norm = norm, # normalized data
raw = raw, # raw data, used to down-weight low-count observations
bg = bg, # expected background counts for every data point in norm
X = safeTME, # safeTME matrix, used by default
cellmerges = safeTME.matches, # safeTME.matches object, used by default
cell_counts = annot$nuclei, # nuclei counts, used to estimate total cells
is_pure_tumor = annot$istumor, # identities of the Tumor segments/observations
n_tumor_clusters = 5) # how many distinct tumor profiles to append to safeTME
str(restils)
```
There are quite a few readouts here. Let's review the important ones:
* beta: the cell abundance scores of the rolled-up/major cell types
* beta.granular: the cell abundance scores of the granular cell types,
corresponding to the columns of the cell profile matrix
* yhat, resids: the fitted values and log2-scale residuals from the
deconvolution fit. Can be used to measure each observation's goodness-of-fit, a
possible QC metric.
* cell.counts, cell.counts.granular: estimated numbers of each cell type,
derived using the nuclei count input
* prop_of_nontumor: the beta matrix rescaled to give the proportions of
non-tumor cells in each observation.
* X: the cell profile matrix used, including newly-derived tumor-specific
columns.
To illustrate the derivation of tumor profiles, let's look at the cell profile
matrix output by spatialdecon:
```{r shownewX, fig.height=5, fig.width=8, fig.cap = "safeTME merged with newly-derived tumor profiles"}
heatmap(sweep(restils$X, 1, apply(restils$X, 1, max), "/"),
labRow = NA, margins = c(10, 5))
```
Note the new tumor-specific columns.
Finally, let's compare deconvolution results from basic vs. the advanced setting
with tumor profiles appended (just for a few cell types):
```{r compareresults, fig.height=6, fig.width=8, fig.cap = "Cell abundance estimates with and without modelling tumor profiles"}
par(mfrow = c(2, 3))
par(mar = c(5,7,2,1))
for (i in seq_len(6)) {
cell = rownames(res$beta)[i]
plot(res$beta[cell, ], restils$beta.granular[cell, ],
xlab = paste0(cell, " score under basic setting"),
ylab = paste0(cell, " score when tumor\ncells are modelled"),
pch = 16,
col = c(rgb(0,0,1,0.5), rgb(1,0,0,0.5))[1 + annot$istumor],
xlim = range(c(res$beta[cell, ], restils$beta.granular[cell, ])),
ylim = range(c(res$beta[cell, ], restils$beta.granular[cell, ])))
abline(0,1)
if (i == 1) {
legend("topleft", pch = 16, col = c(rgb(0,0,1,0.5), rgb(1,0,0,0.5)),
legend = c("microenv.", "tumor"))
}
}
```
So the impact of modelling tumor is two-fold:
* Estimated immune cell content in tumor segments is driven down all the way, or
almost all the way, to 0.
* Estimated immune cell abundance in microenvironment segments in suppressed, as
part of the gene expression is attributed to cancer cells instead of immune
cells.
### Plotting deconvolution results
The SpatialDecon package contains two specialized plotting functions, and a
default color palette for the safeTME matrix.
The first function is "TIL_barplot", which is just a convenient way of drawing
barplots of cell type abundance.
```{r barplot, fig.width=9, fig.height=6, fig.cap="Barplots of TIL abundance"}
# For reference, show the TILs color data object used by the plotting functions
# when safeTME has been used:
data("cellcols")
cellcols
# show just the TME segments, since that's where the immune cells are:
layout(mat = (matrix(c(1, 2), 1)), widths = c(7, 3))
TIL_barplot(restils$cell.counts$cell.counts, draw_legend = TRUE,
cex.names = 0.5)
# or the proportions of cells:
TIL_barplot(restils$prop_of_nontumor[, annot$AOI.name == "TME"],
draw_legend = TRUE, cex.names = 0.75)
```
The second function is "florets", used for plotting cell abundances atop some
2-D projection.
Here, we'll plot cell abundances atop the first 2 principal components of the
data:
```{r florets, fig.width=8, fig.height=6, fig.cap = "TIL abundance plotted on PC space"}
# PCA of the normalized data:
pc = prcomp(t(log2(pmax(norm, 1))))$x[, c(1, 2)]
# run florets function:
par(mar = c(5,5,1,1))
layout(mat = (matrix(c(1, 2), 1)), widths = c(6, 2))
florets(x = pc[, 1], y = pc[, 2],
b = restils$beta, cex = 2,
xlab = "PC1", ylab = "PC2")
par(mar = c(0,0,0,0))
frame()
legend("center", fill = cellcols[rownames(restils$beta)],
legend = rownames(restils$beta), cex = 0.7)
```
So we can see that PC1 roughly tracks many vs. few immune cells, and PC2 tracks
the relative abundance of lymphoid/myeloid populations.
### Other functions
The SpatialDecon library includes several helpful functions for further
analysis/fine-tuning of deconvolution results.
#### Combining cell types:
When two cell types are too similar, the estimation of their abundances becomes
unstable. However, their sum can still be estimated easily.
The function "collapseCellTypes" takes a deconvolution results object and
collapses any colsely-related cell types you tell it to:
```{r collapse, fig.width=5, fig.height=5, fig.cap="Cell abundance estimates with related cell types collapsed"}
matching = list()
matching$myeloid = c( "macrophages", "monocytes", "mDCs")
matching$T.NK = c("CD4.T.cells","CD8.T.cells", "Treg", "NK")
matching$B = c("B")
matching$mast = c("mast")
matching$neutrophils = c("neutrophils")
matching$stroma = c("endothelial.cells", "fibroblasts")
collapsed = collapseCellTypes(fit = restils,
matching = matching)
heatmap(collapsed$beta, cexRow = 0.85, cexCol = 0.75)
```
#### Inferring an expression profile for a cell type omitted from the profile matrix
Sometimes a cell profile matrix will omit a cell type you know to be present in
a tissue.
If your data includes any regions that are purely this unmodelled cell type -
e.g. because you've used the GeoMx platform's segmentation capability to
specifically select them - then you can infer a profile for that cell type and
merge it with your cell profile matrix.
The algorithm clusters all the observations you designate as purely the
unmodelled cell type, and collapses those clusters into as many profiles of that
cell type as you wish. For cancer cell, it may be appropriate to specify 10 or
more clusters; for highly regular healthy cells, one cluster may suffice.
(Note: this functionality can also be run within the spatialdecon function, as
is demonstrated further above.)
```{r appendtumor, fig.width = 10, fig.height= 5, fig.cap = "safeTME merged with newly-derived tumor profiles"}
pure.tumor.ids = annot$AOI.name == "Tumor"
safeTME.with.tumor = mergeTumorIntoX(norm = norm,
bg = bg,
pure_tumor_ids = pure.tumor.ids,
X = safeTME,
K = 3)
heatmap(sweep(safeTME.with.tumor, 1, apply(safeTME.with.tumor, 1, max), "/"),
labRow = NA, margins = c(10, 5))
```
#### Reverse deconvolution
Once cell type abundance has been estimated, we can flip the deconvolution
around, modelling the expression data as a function of cell abundances, and
thereby deriving:
1. Estimated expression of each gene in each cell type. (Including for genes
not present in your cell profile matrix)
2. Fitted expression values for each gene based on cell mixing.
3. Residuals of each gene: how does their expression compare to what cell mixing
would predict?
4. Two metrics of how well genes are predicted by/ redundant with cell mixing:
correlation between observed and fitted expression, and residual SD.
The function "reversedecon" runs this model.
```{r reverse, fig.height=6, fig.width=6, fig.cap="Residuals from reverseDecon"}
rdecon = reverseDecon(norm = norm,
beta = res$beta)
str(rdecon)
# look at the residuals:
heatmap(pmax(pmin(rdecon$resids, 2), -2))
```
```{r reverse2, fig.height=6, fig.width=6, fig.cap="Genes' dependency on cell mixing"}
# look at the two metrics of goodness-of-fit:
plot(rdecon$cors, rdecon$resid.sd, col = 0)
showgenes = c("CXCL14", "LYZ", "NKG7")
text(rdecon$cors[setdiff(names(rdecon$cors), showgenes)],
rdecon$resid.sd[setdiff(names(rdecon$cors), showgenes)],
setdiff(names(rdecon$cors), showgenes), cex = 0.5)
text(rdecon$cors[showgenes], rdecon$resid.sd[showgenes],
showgenes, cex = 0.75, col = 2)
```
From the above plot, we can see that genes like CXCL14 vary independently of
cell mixing, genes like LYZ are correlated with cell mixing but still have
variable expression, and genes like NKG7 serve as nothing but obtuse readouts
of cell mixing.
### Session Info
```{r sessioninfo}
sessionInfo()
```