16 Seurat
Seurat was originally developed as a clustering
tool for scRNA-seq data, however in the last few years the focus of the package
has become less specific and at the moment Seurat is a popular R package that
can perform QC, analysis, and exploration of scRNA-seq data, i.e. many of the
tasks covered in this course.
Note We recommend using Seurat for datasets with more than \(5000\) cells. For smaller dataset a good alternative will be SC3.
Note In this chapter we use an exact copy of this tutorial.
16.1 Setup the Seurat Object
We will be analyzing the a dataset of Peripheral Blood Mononuclear Cells (PBMC) freely available from 10X Genomics. There are 2,700 single cells that were sequenced on the Illumina NextSeq 500. The raw data can be found here.
We start by reading in the data. All features in Seurat have been configured to work with sparse matrices which results in significant memory and speed savings for Drop-seq/inDrop/10x data.
library(Seurat)
library(dplyr)
library(cowplot)
# Load the PBMC dataset
pbmc.data <- Read10X(data.dir = "data/pbmc3k_filtered_gene_bc_matrices/hg19/")
# Examine the memory savings between regular and sparse matrices
dense.size <- object.size(x = as.matrix(x = pbmc.data))
dense.size
sparse.size <- object.size(x = pbmc.data)
sparse.size
dense.size/sparse.size
# Initialize the Seurat object with the raw (non-normalized data). Keep all
# genes expressed in >= 3 cells (~0.1% of the data). Keep all cells with at
# least 200 detected genes
pbmc <- CreateSeuratObject(counts = pbmc.data, min.cells = 3, min.features = 200, project = "10X_PBMC", assay = "RNA")16.2 Standard pre-processing workflow
The steps below encompass the standard pre-processing workflow for scRNA-seq data in Seurat. These represent the creation of a Seurat object, the selection and filtration of cells based on QC metrics, data normalization and scaling, and the detection of highly variable genes.
16.3 QC and selecting cells for further analysis
While the CreateSeuratObject imposes a basic minimum gene-cutoff, you may want
to filter out cells at this stage based on technical or biological parameters.
Seurat allows you to easily explore QC metrics and filter cells based on any
user-defined criteria. In the example below, we visualize gene and molecule
counts, plot their relationship, and exclude cells with a clear outlier number
of genes detected as potential multiplets. Of course this is not a guaranteed
method to exclude cell doublets, but we include this as an example of filtering
user-defined outlier cells. We also filter cells based on the percentage of
mitochondrial genes present.
# The number of genes and UMIs (nGene and nUMI) are automatically calculated
# for every object by Seurat. For non-UMI data, nUMI represents the sum of
# the non-normalized values within a cell We calculate the percentage of
# mitochondrial genes here and store it in percent.mito using AddMetaData.
# We use [email protected] since this represents non-transformed and
# non-log-normalized counts The % of UMI mapping to MT-genes is a common
# scRNA-seq QC metric.
mito.genes <- grep(pattern = "^MT-", x = rownames(pbmc@assays[["RNA"]]), value = TRUE)
percent.mito <- Matrix::colSums(pbmc@assays[["RNA"]][mito.genes, ])/Matrix::colSums(pbmc@assays[["RNA"]])
# AddMetaData adds columns to [email protected], and is a great place to
# stash QC stats
#Seurat v2 function, but shows compatibility in Seurat v3
pbmc <- AddMetaData(object = pbmc, metadata = percent.mito, col.name = "percent.mito")
#in case the above function does not work simply do:
pbmc$percent.mito <- percent.mito
VlnPlot(object = pbmc, features = c("nFeature_RNA", "nCount_RNA", "percent.mito"), ncol = 3)# GenePlot is typically used to visualize gene-gene relationships, but can
# be used for anything calculated by the object, i.e. columns in
# [email protected], PC scores etc. Since there is a rare subset of cells
# with an outlier level of high mitochondrial percentage and also low UMI
# content, we filter these as well
par(mfrow = c(1, 2))
FeatureScatter(object = pbmc, feature1 = "nCount_RNA", feature2 = "percent.mito")
FeatureScatter(object = pbmc, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")# We filter out cells that have unique gene counts (nFeature_RNA) over 2,500 or less than
# 200 Note that > and < are used to define a'gate'.
#-Inf and Inf should be used if you don't want a lower or upper threshold.
pbmc <- subset(x = pbmc, subset = nFeature_RNA > 200 & nFeature_RNA < 2500 & percent.mito > -Inf & percent.mito < 0.05 )16.4 Normalizing the data
After removing unwanted cells from the dataset, the next step is to normalize the data. By default, we employ a global-scaling normalization method “LogNormalize” that normalizes the gene expression measurements for each cell by the total expression, multiplies this by a scale factor (10,000 by default), and log-transforms the result.
16.5 Detection of variable genes across the single cells
Seurat calculates highly variable genes and focuses on these for downstream
analysis. FindVariableGenes calculates the average expression and dispersion
for each gene, places these genes into bins, and then calculates a z-score for
dispersion within each bin. This helps control for the relationship between
variability and average expression. This function is unchanged from (Macosko et
al.), but new methods for variable gene expression identification are coming
soon. We suggest that users set these parameters to mark visual outliers on the
dispersion plot, but the exact parameter settings may vary based on the data
type, heterogeneity in the sample, and normalization strategy. The parameters
here identify ~2,000 variable genes, and represent typical parameter settings
for UMI data that is normalized to a total of 1e4 molecules.
To view the output of the FindVariableFeatures output we use this function. The genes appear not to be stored in the object, but can be accessed this way.
16.6 Scaling the data and removing unwanted sources of variation
Your single cell dataset likely contains ‘uninteresting’ sources of variation. This could include not only technical noise, but batch effects, or even biological sources of variation (cell cycle stage). As suggested in Buettner et al, NBT, 2015, regressing these signals out of the analysis can improve downstream dimensionality reduction and clustering. To mitigate the effect of these signals, Seurat constructs linear models to predict gene expression based on user-defined variables. The scaled z-scored residuals of these models are stored in the scale.data slot, and are used for dimensionality reduction and clustering.
We can regress out cell-cell variation in gene expression driven by batch (if applicable), cell alignment rate (as provided by Drop-seq tools for Drop-seq data), the number of detected molecules, and mitochondrial gene expression. For cycling cells, we can also learn a ‘cell-cycle’ score (see example here) and regress this out as well. In this simple example here for post-mitotic blood cells, we regress on the number of detected molecules per cell as well as the percentage mitochondrial gene content.
Seurat v2.0 implements this regression as part of the data scaling process.
Therefore, the RegressOut function has been deprecated, and replaced with the
vars.to.regress argument in ScaleData.
16.7 Perform linear dimensional reduction
–> refered to Seurat v2: Next we perform PCA on the scaled data. By default,
the genes in [email protected] are used as input, but can be defined using
pc.genes. We have typically found that running dimensionality reduction on
highly variable genes can improve performance. However, with UMI data -
particularly after regressing out technical variables, we often see that PCA
returns similar (albeit slower) results when run on much larger subsets of
genes, including the whole transcriptome.
–> refered to Seurat v3 (latest): high variable features are accessed through the function HVFInfo(object). Despite RunPCA has a features argument where to specify the features to compute PCA on, I’ve been modifying its values and the output PCA graph has always the same dimensions, indicating that the provided genes in the features argument are not exactly the ones used to compute PCA. Wether the function gets the HVG directly or does not take them into account, I don’t know.
–> refered to Seurat v2: Seurat provides several useful ways of visualizing
both cells and genes that define the PCA, including PrintPCA, VizPCA,
PCAPlot, and PCHeatmap
–> refered to Seurat v3 (latest): Seurat v3 provides functions for visualizing: - PCA - PCA plot coloured by a quantitative feature - Scatter plot across single cells - Scatter plot across individual features - Variable Feature Plot - Violin and Ridge plots - Heatmaps
# Dimensional reduction plot, with cells colored by a quantitative feature
FeaturePlot(object = pbmc, features = "MS4A1")# Scatter plot across single cells, replaces GenePlot
FeatureScatter(object = pbmc, feature1 = "MS4A1", feature2 = "PC_1")
FeatureScatter(object = pbmc, feature1 = "MS4A1", feature2 = "CD3D")# Scatter plot across individual features, repleaces CellPlot
CellScatter(object = pbmc, cell1 = "AGTCTACTAGGGTG", cell2 = "CACAGATGGTTTCT")# Violin and Ridge plots
VlnPlot(object = pbmc, features = c("LYZ", "CCL5", "IL32"))
RidgePlot(object = pbmc, feature = c("LYZ", "CCL5", "IL32"))In particular DimHeatmap allows for easy exploration of the primary sources of
heterogeneity in a dataset, and can be useful when trying to decide which PCs to
include for further downstream analyses. Both cells and genes are ordered
according to their PCA scores. Setting cells.use to a number plots the ‘extreme’
cells on both ends of the spectrum, which dramatically speeds plotting for large
datasets. Though clearly a supervised analysis, we find this to be a valuable
tool for exploring correlated gene sets.
ProjectPCA function is no loger available in Seurat 3.0.
16.8 Determine statistically significant principal components
To overcome the extensive technical noise in any single gene for scRNA-seq data, Seurat clusters cells based on their PCA scores, with each PC essentially representing a ‘metagene’ that combines information across a correlated gene set. Determining how many PCs to include downstream is therefore an important step.
In Macosko et al, we implemented a resampling test inspired by the jackStraw procedure. We randomly permute a subset of the data (1% by default) and rerun PCA, constructing a ‘null distribution’ of gene scores, and repeat this procedure. We identify ‘significant’ PCs as those who have a strong enrichment of low p-value genes.
# NOTE: This process can take a long time for big datasets, comment out for
# expediency. More approximate techniques such as those implemented in
# PCElbowPlot() can be used to reduce computation time
pbmc <- JackStraw(object = pbmc, reduction = "pca", dims = 20, num.replicate = 100, prop.freq = 0.1, verbose = FALSE)The JackStrawPlot function provides a visualization tool for comparing the
distribution of p-values for each PC with a uniform distribution (dashed line).
‘Significant’ PCs will show a strong enrichment of genes with low p-values
(solid curve above the dashed line). In this case it appears that PCs 1-10 are
significant.
A more ad hoc method for determining which PCs to use is to look at a plot of
the standard deviations of the principle components and draw your cutoff where
there is a clear elbow in the graph. This can be done with ElbowPlot. In this
example, it looks like the elbow would fall around PC 5.
PC selection – identifying the true dimensionality of a dataset – is an important step for Seurat, but can be challenging/uncertain for the user. We therefore suggest these three approaches to consider. The first is more supervised, exploring PCs to determine relevant sources of heterogeneity, and could be used in conjunction with GSEA for example. The second implements a statistical test based on a random null model, but is time-consuming for large datasets, and may not return a clear PC cutoff. The third is a heuristic that is commonly used, and can be calculated instantly. In this example, all three approaches yielded similar results, but we might have been justified in choosing anything between PC 7-10 as a cutoff. We followed the jackStraw here, admittedly buoyed by seeing the PCHeatmap returning interpretable signals (including canonical dendritic cell markers) throughout these PCs. Though the results are only subtly affected by small shifts in this cutoff (you can test below), we strongly suggest always explore the PCs they choose to include downstream.
16.9 Cluster the cells
Seurat now includes an graph-based clustering approach compared to (Macosko et al.). Importantly, the distance metric which drives the clustering analysis (based on previously identified PCs) remains the same. However, our approach to partioning the cellular distance matrix into clusters has dramatically improved. Our approach was heavily inspired by recent manuscripts which applied graph-based clustering approaches to scRNA-seq data SNN-Cliq, Xu and Su, Bioinformatics, 2015 and CyTOF data PhenoGraph, Levine et al., Cell, 2015. Briefly, these methods embed cells in a graph structure - for example a K-nearest neighbor (KNN) graph, with edges drawn between cells with similar gene expression patterns, and then attempt to partition this graph into highly interconnected ‘quasi-cliques’ or ‘communities’. As in PhenoGraph, we first construct a KNN graph based on the euclidean distance in PCA space, and refine the edge weights between any two cells based on the shared overlap in their local neighborhoods (Jaccard similarity). To cluster the cells, we apply modularity optimization techniques such as the Louvain algorithm (default) or SLM SLM, Blondel et al., Journal of Statistical Mechanics, to iteratively group cells together, with the goal of optimizing the standard modularity function.
The FindClusters function implements the procedure, and contains a resolution
parameter that sets the ‘granularity’ of the downstream clustering, with
increased values leading to a greater number of clusters. We find that setting
this parameter between 0.6-1.2 typically returns good results for single cell
datasets of around 3K cells. Optimal resolution often increases for larger
datasets. Latest clustering results will be stored in object metadata under
seurat_clusters.
First calculate k-nearest neighbors and construct the SNN graph
(FindNeighbors), then run FindClusters.
16.10 Run Non-linear dimensional reduction (tSNE)
Seurat continues to use tSNE as a powerful tool to visualize and explore these datasets. While we no longer advise clustering directly on tSNE components, cells within the graph-based clusters determined above should co-localize on the tSNE plot. This is because the tSNE aims to place cells with similar local neighborhoods in high-dimensional space together in low-dimensional space. As input to the tSNE, we suggest using the same PCs as input to the clustering analysis, although computing the tSNE based on scaled gene expression is also supported using the genes.use argument.
16.11 Run UMAP
To visualize the two conditions side-by-side, we can use the split.by argument to show each condition colored by cluster.
pbmc <- RunUMAP(pbmc, reduction = "pca", dims = 1:20)
DimPlot(pbmc, reduction = "umap", split.by = "seurat_clusters")You can save the object at this point so that it can easily be loaded back in without having to rerun the computationally intensive steps performed above, or easily shared with collaborators.
16.12 Finding differentially expressed genes (cluster biomarkers)
Seurat can help you find markers that define clusters via differential
expression. By default, it identifes positive and negative markers of a single
cluster (specified in ident.1), compared to all other cells. FindAllMarkers
automates this process for all clusters, but you can also test groups of
clusters vs. each other, or against all cells.
The min.pct argument requires a gene to be detected at a minimum percentage in
either of the two groups of cells, and the thresh.test argument requires a gene
to be differentially expressed (on average) by some amount between the two
groups. You can set both of these to 0, but with a dramatic increase in time -
since this will test a large number of genes that are unlikely to be highly
discriminatory. As another option to speed up these computations,
max.cells.per.ident can be set. This will downsample each identity class to
have no more cells than whatever this is set to. While there is generally going
to be a loss in power, the speed increases can be significiant and the most
highly differentially expressed genes will likely still rise to the top.
# find all markers of cluster 1
cluster1.markers <- FindMarkers(object = pbmc, ident.1 = 1, min.pct = 0.25)
print(x = head(x = cluster1.markers, n = 5))# find all markers distinguishing cluster 5 from clusters 0 and 3
cluster5.markers <- FindMarkers(object = pbmc, ident.1 = 2, ident.2 = c(0, 3), min.pct = 0.25)
print(x = head(x = cluster5.markers, n = 5))# find markers for every cluster compared to all remaining cells, report
# only the positive ones
pbmc.markers <- FindAllMarkers(object = pbmc, only.pos = TRUE, min.pct = 0.25, thresh.use = 0.25)
pbmc.markers %>% group_by(cluster) %>% top_n(2, avg_logFC)Seurat has several tests for differential expression which can be set with the test.use parameter (see our DE vignette for details). For example, the ROC test returns the ‘classification power’ for any individual marker (ranging from 0 - random, to 1 - perfect).
We include several tools for visualizing marker expression.
• VlnPlot (shows expression probability distributions across clusters),
• and FeaturePlot (visualizes gene expression on a tSNE or PCA plot) are our most commonly used visualizations.
We also suggest exploring:
• RidgePlot,
• CellPlot, and
• DotPlot as additional methods to view your dataset.
DoHeatmap generates an expression heatmap for given cells and genes. In this
case, we are plotting the top 20 markers (or all markers if less than 20) for
each cluster.
16.13 Assigning cell type identity to clusters
Fortunately in the case of this dataset, we can use canonical markers to easily match the unbiased clustering to known cell types.
current.cluster.ids <- c(0, 1, 2, 3, 4, 5, 6, 7)
new.cluster.ids <- c("CD4 T cells", "CD14+ Monocytes", "B cells", "CD8 T cells", "FCGR3A+ Monocytes", "NK cells", "Dendritic cells", "Megakaryocytes")
pbmc@active.ident <- plyr::mapvalues(x = pbmc@active.ident, from = current.cluster.ids, to = new.cluster.ids)
DimPlot(object = pbmc, reduction = "tsne", do.label = TRUE, pt.size = 0.5)16.14 Further subdivisions within cell types
If you perturb some of our parameter choices above (for example, setting
resolution=0.8 or changing the number of PCs), you might see the CD4 T cells
subdivide into two groups. You can explore this subdivision to find markers
separating the two T cell subsets. However, before reclustering (which will
overwrite object@ident), we can stash our renamed identities to be easily
recovered later.
# First lets stash our identities for later
pbmc <- StashIdent(object = pbmc, save.name = "ClusterNames_0.6")
# Note that if you set save.snn=T above, you don't need to recalculate the
# SNN, and can simply put: pbmc <- FindClusters(pbmc,resolution = 0.8)
pbmc <- FindClusters(object = pbmc, reduction.type = "pca", dims.use = 1:10, resolution = 0.8, print.output = FALSE)# Demonstration of how to plot two tSNE plots side by side, and how to color
# points based on different criteria
plot1 <- DimPlot(object = pbmc, reduction = "tsne", do.return = TRUE, no.legend = TRUE, do.label = TRUE)
plot2 <- DimPlot(object = pbmc, reduction = "tsne", do.return = TRUE, group.by = "ClusterNames_0.6", no.legend = TRUE, do.label = TRUE)
plot_grid(plot1, plot2)# Find discriminating markers
tcell.markers <- FindMarkers(object = pbmc, ident.1 = 0, ident.2 = 1)
# Most of the markers tend to be expressed in C1 (i.e. S100A4). However, we
# can see that CCR7 is upregulated in C0, strongly indicating that we can
# differentiate memory from naive CD4 cells. cols.use demarcates the color
# palette from low to high expression
FeaturePlot(object = pbmc, features = c("S100A4", "CCR7"), cols = c("green", "blue"))The memory/naive split is bit weak, and we would probably benefit from looking at more cells to see if this becomes more convincing. In the meantime, we can restore our old cluster identities for downstream processing.