1 Introduction

Spatial transcriptomics (ST), named Method of the Year 2020 by Nature Methods in 2020, is a powerful and widely-used experimental method for profiling genome-wide gene expression across a tissue. In a typical ST experiment, fresh-frozen (or FFPE) tissue is sectioned and placed onto a slide containing spots, with each spot containing millions of capture oligonucleotides with spatial barcodes unique to that spot. The tissue is imaged, typically via Hematoxylin and Eosin (H&E) staining. Following imaging, the tissue is permeabilized to release mRNA which then binds to the capture oligonucleotides, generating a cDNA library consisting of transcripts bound by barcodes that preserve spatial information. Data from an ST experiment consists of the tissue image coupled with RNA-sequencing data collected from each spot. A first step in processing ST data is tissue detection, where spots on the slide containing tissue are distinguished from background spots without tissue. Unique molecular identifier (UMI) counts at each spot containing tissue are then used in downstream analyses.

Ideally, a gene-specific UMI at a given spot would represent expression of that gene at that spot, and spots without tissue would show no (or few) UMIs. This is not the case in practice. Messenger RNA bleed from nearby spots causes substantial contamination of UMI counts, an artifact we refer to as spot swapping. On average, we observe that more than 30% of UMIs at a tissue spot did not originate from this spot, but from other spots contaminating it. Spot swapping confounds downstream inferences including normalization, marker gene-based annotation, differential expression and cell type decomposition.

We developed SpotClean to adjust for the effects of spot swapping in ST experiments. SpotClean is able to measure the per-spot contamination rates in observed data and decontaminate gene expression levels, thus increases the sensitivity and precision of downstream analyses. Our package SpotClean is built based on 10x Visium spatial transcriptomics experiments, currently the most widely-used commercial protocol, providing functions to load raw spatial transcriptomics data from 10x Space Ranger outputs, decontaminate the spot swapping effect, estimate contamination levels, visualize expression profiles and spot labels on the slide, and connect with other widely-used packages for further analyses. SpotClean can be potentially extended to other spatial transcriptomics data as long as the gene expression data in both tissue and background regions are available. SpotClean is compatible with the SpatialExperiment class and supports downstream analyses via Seurat.

As a computational tool for analyzing spatial transcriptomics data, SpotClean has been submitted to Bioconductor, making the R package well-documented and well-maintained for improved reproducibility and user experience.

2 Quick Start

2.1 Installation

Install from Bioconductor:

if (!requireNamespace("BiocManager", quietly = TRUE))


Load package after installation:


2.2 Short Demo

Here we quickly demonstrate the general SpotClean workflow on the built-in example data. A step-by-step illustration can be found in the next section.

Note: codes in this chunk are only for illustrative purpose and are not runnable. Runnable codes can be found in the next section.

# Not run

# Load 10x Visium data
mbrain_raw <- read10xRaw("/path/to/matrix/folder")
mbrain_slide_info <- read10xSlide("/path/to/tissue/csv", 

# Visualize raw data
mbrain_obj <- createSlide(count_mat = mbrain_raw, 
                          slide_info = mbrain_slide_info)
visualizeSlide(slide_obj = mbrain_obj)

# Decontaminate raw data
decont_obj <- spotclean(mbrain_obj)

# Visualize decontaminated gene

# Visualize the estimated per-spot contamination rate
                 logged = FALSE, legend_title = "contamination rate",
                 legend_range = c(0,1))

# (Optionally) Transform to Seurat object for downstream analyses
seurat_obj <- convertToSeurat(decont_obj,image_dir = "/path/to/spatial/folder")

2.3 Working with the SpatialExperiment class

SpatialExperiment is another widely used S4 class for storing ST data, including the count matrix, cell and gene-level metadata, and tissue image. spotclean() can be directly applied to SpatialExperiment objects constructed from SpatialExperiment::read10xVisium(), which reads in the raw data from 10x Visium Space Ranger output. The visualization functions in our package currently do not support SpatialExperiment class, but there are alternative options such as ggspavis.

# Not run
slide_obj <- read10xVisium(samples = "/path/to/spaceranger/output/", 
                         data = "raw") # must specify data = "raw"
decont_obj <- spotclean(slide_obj)

2.4 Running Speed

The computational speed is related to the size and structure of input datasets, mainly driven by the number of tissue spots. SpotClean does not require parallel computation, and thus does not use up too many CPU or memory resources. As a reference, SpotClean running on a medium-size dataset (around 30,000 genes and 2,000 tissue spots) under default gene filtering takes less than 15 minutes.

2.5 Situations you should think twice about before applying SpotClean

  • Too many tissue spots (not enough background spots) While the observed data is a single matrix with a fixed number of columns (spots), the number of unknown parameters is proportional to the number of tissue spots. In the extreme case where all spots are covered by tissue, we have more unknown parameters than observed data values. In this case the contaminated expressions are confounded with true expressions, and SpotClean estimation becomes unreliable. We recommend that the input data have at least 25% spots not occupied by tissue, so that SpotClean has enough information from background spots to estimate contamination.

  • Lowly-expressed genes Lowly-expressed genes typically contain relatively less information and relatively more noise than highly-expressed genes. SpotClean by default only keeps highly-expressed or highly-variable genes for decontamination (or both). It can be forced to run on manually-specified lowly-expressed genes. However, even in this case, expression for the lowly-expressed genes is typically not changed very much. Given the high sparsity in most lowly expressed genes, there is not enough information available to confidently reassign UMIs in most cases. However, we do not filter genes by sparsity because there can be interesting genes highly concentrated in a small tissue region. In cases like this, SpotClean is effective at adjusting for spot swapping in these regions. If the defaults are not appropriate, users can either adjust the expression cutoffs or manually specify genes to decontaminate.

  • Inference based on sequencing depth SpotClean reassigns bled-out UMIs to their tissue spots of origin which changes the estimated sequencing depth of tissue spots after decontamination, since most estimations of sequencing depth rely on total expressions at every spot. As a result, decontamination can be considered as another type of normalization and might conflict with existing sequencing depth normalization methods.

3 Detailed Steps

3.1 Load count matrix and slide information from 10x Space Ranger output

Currently, the most widely-used spatial transcriptomics protocol is 10x Visium. Public 10x datasets can be found here. In this vignette, we illustrate the usage of SpotClean using the V1_Adult_Mouse_Brain Visium data.

Two parts of 10x Space Ranger output files are required as input to SpotClean: the raw gene-by-spot count matrix, and the slide metadata. In this example, you can download and unzip the Feature / cell matrix (raw) and Spatial imaging data from V1_Adult_Mouse_Brain. You will get a folder raw_feature_bc_matrix containing barcodes.tsv.gz, features.tsv.gz, matrix.mtx.gz, and a folder spatial containing aligned_fiducials.jpg, detected_tissue_image.jpg, tissue_hires_image.png, tissue_lowres_image.png, tissue_positions_list.csv, scalefactors_json.json. The former folder contains the raw gene-by-spot count matrix, and the latter contains slide metadata like slide images and spot IDs and positions.

SpotClean provides functions read10xRaw() and read10xSlide() to directly read these 10x Space Ranger output files and generate a gene-by-spot count matrix and slide metadata in R. Here for the purpose of demonstration, we have provided the count matrix and slide metadata with our package. The count matrix is a built-in example object mbrain_raw, which is a subset of the gene-by-spot count matrix from read10xRaw(), containing the top 100 highest expressed genes. The slide metadata, located at extdata/V1_Adult_Mouse_Brain_spatial, contains the three basic files (tissue_positions_list.csv, tissue_lowres_image.png, scalefactors_json.json) to run SpotClean and other associated packages in this vignette.

# load count matrix
## Formal class 'dgCMatrix' [package "Matrix"] with 6 slots
##   ..@ i       : int [1:457217] 0 1 2 3 4 5 6 7 8 9 ...
##   ..@ p       : int [1:4993] 0 99 199 299 399 499 592 692 788 857 ...
##   ..@ Dim     : int [1:2] 100 4992
##   ..@ Dimnames:List of 2
##   .. ..$ : chr [1:100] "Bc1" "Gm42418" "mt-Co3" "mt-Atp6" ...
##   ..@ x       : num [1:457217] 141 266 217 202 106 102 84 50 56 44 ...
##   ..@ factors : list()
# read spatial metadata
spatial_dir <- system.file(file.path("extdata",
                           package = "SpotClean")
## [1] "scalefactors_json.json"    "tissue_lowres_image.png"  
## [3] "tissue_positions_list.csv"
mbrain_slide_info <- read10xSlide(tissue_csv_file=file.path(spatial_dir,
             tissue_img_file = file.path(spatial_dir,
             scale_factor_file = file.path(spatial_dir,
## List of 2
##  $ slide:'data.frame':   4992 obs. of  8 variables:
##   ..$ tissue  : Factor w/ 2 levels "0","1": 1 1 1 1 1 1 1 1 1 1 ...
##   ..$ row     : int [1:4992] 0 1 0 1 0 1 0 1 0 1 ...
##   ..$ col     : int [1:4992] 0 1 2 3 4 5 6 7 8 9 ...
##   ..$ imagerow: num [1:4992] 63.8 70 63.8 70 63.8 ...
##   ..$ imagecol: num [1:4992] 61.8 65.3 68.8 72.3 75.8 ...
##   ..$ height  : int [1:4992] 600 600 600 600 600 600 600 600 600 600 ...
##   ..$ width   : int [1:4992] 576 576 576 576 576 576 576 576 576 576 ...
##  $ grob :List of 12
##   ..$ raster     : 'raster' chr [1:600, 1:576] "#81837E" "#80837E" "#81837E" "#81837E" ...
##   ..$ x          : 'simpleUnit' num 0.5npc
##   .. ..- attr(*, "unit")= int 0
##   ..$ y          : 'simpleUnit' num 0.5npc
##   .. ..- attr(*, "unit")= int 0
##   ..$ width      : 'simpleUnit' num 1npc
##   .. ..- attr(*, "unit")= int 0
##   ..$ height     : 'simpleUnit' num 1npc
##   .. ..- attr(*, "unit")= int 0
##   ..$ just       : chr "centre"
##   ..$ hjust      : NULL
##   ..$ vjust      : NULL
##   ..$ interpolate: logi TRUE
##   ..$ name       : chr "GRID.rastergrob.11"
##   ..$ gp         : list()
##   .. ..- attr(*, "class")= chr "gpar"
##   ..$ vp         : NULL
##   ..- attr(*, "class")= chr [1:3] "rastergrob" "grob" "gDesc"

3.2 Create the slide object

In the following, we will show the SpotClean workflow using the built-in example data.

We combine count matrix and slide metadata together as one single slide object with class SummarizedExperiment:

slide_obj <- createSlide(mbrain_raw, mbrain_slide_info)
## class: SummarizedExperiment 
## dim: 100 4992 
## metadata(2): slide grob
## assays(1): raw
## rownames(100): Bc1 Gm42418 ... Rps25 Rps20
## rowData names(0):
## colData names(0):

As shown above, the raw count matrix is stored in the raw assay slot, and slide and image information are stored in slide and grob metadata slots.

3.3 Visualize the slide object

Our package provides multiple visualization functions in the 2-D slide space. Function visualizeSlide() shows the input slide imaging file (if given) in R: