# Unfiltered human PBMCs (10X Genomics)
## Introduction
Here, we describe a brief analysis of the peripheral blood mononuclear cell (PBMC) dataset from 10X Genomics [@zheng2017massively].
The data are publicly available from the [10X Genomics website](https://support.10xgenomics.com/single-cell-gene-expression/datasets/2.1.0/pbmc4k),
from which we download the raw gene/barcode count matrices, i.e., before cell calling from the _CellRanger_ pipeline.
## Data loading
``` r
library(DropletTestFiles)
raw.path <- getTestFile("tenx-2.1.0-pbmc4k/1.0.0/raw.tar.gz")
out.path <- file.path(tempdir(), "pbmc4k")
untar(raw.path, exdir=out.path)
library(DropletUtils)
fname <- file.path(out.path, "raw_gene_bc_matrices/GRCh38")
sce.pbmc <- read10xCounts(fname, col.names=TRUE)
```
``` r
library(scater)
rownames(sce.pbmc) <- uniquifyFeatureNames(
rowData(sce.pbmc)$ID, rowData(sce.pbmc)$Symbol)
library(EnsDb.Hsapiens.v86)
location <- mapIds(EnsDb.Hsapiens.v86, keys=rowData(sce.pbmc)$ID,
column="SEQNAME", keytype="GENEID")
```
## Quality control
We perform cell detection using the `emptyDrops()` algorithm, as discussed in [Advanced Section 7.2](http://bioconductor.org/books/3.21/OSCA.advanced/droplet-processing.html#qc-droplets).
``` r
set.seed(100)
e.out <- emptyDrops(counts(sce.pbmc))
sce.pbmc <- sce.pbmc[,which(e.out$FDR <= 0.001)]
```
``` r
unfiltered <- sce.pbmc
```
We use a relaxed QC strategy and only remove cells with large mitochondrial proportions, using it as a proxy for cell damage.
This reduces the risk of removing cell types with low RNA content, especially in a heterogeneous PBMC population with many different cell types.
``` r
stats <- perCellQCMetrics(sce.pbmc, subsets=list(Mito=which(location=="MT")))
high.mito <- isOutlier(stats$subsets_Mito_percent, type="higher")
sce.pbmc <- sce.pbmc[,!high.mito]
```
``` r
summary(high.mito)
```
```
## Mode FALSE TRUE
## logical 3951 313
```
``` r
colData(unfiltered) <- cbind(colData(unfiltered), stats)
unfiltered$discard <- high.mito
gridExtra::grid.arrange(
plotColData(unfiltered, y="sum", colour_by="discard") +
scale_y_log10() + ggtitle("Total count"),
plotColData(unfiltered, y="detected", colour_by="discard") +
scale_y_log10() + ggtitle("Detected features"),
plotColData(unfiltered, y="subsets_Mito_percent",
colour_by="discard") + ggtitle("Mito percent"),
ncol=2
)
```
(\#fig:unref-unfiltered-pbmc-qc)Distribution of various QC metrics in the PBMC dataset after cell calling. Each point is a cell and is colored according to whether it was discarded by the mitochondrial filter.
``` r
plotColData(unfiltered, x="sum", y="subsets_Mito_percent",
colour_by="discard") + scale_x_log10()
```
(\#fig:unref-unfiltered-pbmc-mito)Proportion of mitochondrial reads in each cell of the PBMC dataset compared to its total count.
## Normalization
``` r
library(scran)
set.seed(1000)
clusters <- quickCluster(sce.pbmc)
sce.pbmc <- computeSumFactors(sce.pbmc, cluster=clusters)
sce.pbmc <- logNormCounts(sce.pbmc)
```
``` r
summary(sizeFactors(sce.pbmc))
```
```
## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 0.007 0.717 0.878 1.000 1.102 11.504
```
``` r
plot(librarySizeFactors(sce.pbmc), sizeFactors(sce.pbmc), pch=16,
xlab="Library size factors", ylab="Deconvolution factors", log="xy")
```
(\#fig:unref-unfiltered-pbmc-norm)Relationship between the library size factors and the deconvolution size factors in the PBMC dataset.
## Variance modelling
``` r
set.seed(1001)
dec.pbmc <- modelGeneVarByPoisson(sce.pbmc)
top.pbmc <- getTopHVGs(dec.pbmc, prop=0.1)
```
``` r
plot(dec.pbmc$mean, dec.pbmc$total, pch=16, cex=0.5,
xlab="Mean of log-expression", ylab="Variance of log-expression")
curfit <- metadata(dec.pbmc)
curve(curfit$trend(x), col='dodgerblue', add=TRUE, lwd=2)
```
(\#fig:unref-unfiltered-pbmc-var)Per-gene variance as a function of the mean for the log-expression values in the PBMC dataset. Each point represents a gene (black) with the mean-variance trend (blue) fitted to simulated Poisson counts.
## Dimensionality reduction
``` r
set.seed(10000)
sce.pbmc <- denoisePCA(sce.pbmc, subset.row=top.pbmc, technical=dec.pbmc)
set.seed(100000)
sce.pbmc <- runTSNE(sce.pbmc, dimred="PCA")
set.seed(1000000)
sce.pbmc <- runUMAP(sce.pbmc, dimred="PCA")
```
We verify that a reasonable number of PCs is retained.
``` r
ncol(reducedDim(sce.pbmc, "PCA"))
```
```
## [1] 9
```
## Clustering
``` r
g <- buildSNNGraph(sce.pbmc, k=10, use.dimred = 'PCA')
clust <- igraph::cluster_walktrap(g)$membership
colLabels(sce.pbmc) <- factor(clust)
```
``` r
table(colLabels(sce.pbmc))
```
```
##
## 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
## 202 590 523 780 55 359 126 753 46 151 144 77 82 25 17 21
```
``` r
plotTSNE(sce.pbmc, colour_by="label")
```
(\#fig:unref-unfiltered-pbmc-tsne)Obligatory $t$-SNE plot of the PBMC dataset, where each point represents a cell and is colored according to the assigned cluster.
