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1. Introduction and dataset

In this vignette, we will use the omnideconv package to deconvolve a bulk RNA-seq dataset from 24 breast cancer patients with two different methods (DWLS and BayesPrism). The datasets are from a recent breast cancer study (Wu et al. 2021). This study provides access to a primary-tumor single cell RNA-seq (scRNA-seq) dataset from 26 breast cancer patients across three major cancer subtypes (ER+, HER2+, TNBC). The dataset includes cell-type annotation for three resolution levels. In addition, the data includes bulk RNA-seq sequencing for 24 of the patients. In this chapter, we use multi-sample, breast-cancer scRNA-seq atlas (100,064 cells) as a reference to train the methods for the deconvolution of the bulk RNA-seq samples. The single-cell and bulk RNA-seq data is deposited on GEO, under accession number: GSE176078. The single cell data comes with the author’s cell type annotations.
We will need to download and unzip the datasets (GSE176078_Wu_etal_2021_BRCA_scRNASeq.tar.gz, GSE176078_Wu_etal_2021_bulkRNAseq_raw_counts.txt.gz), and store them in the working directory. For this example analysis, we will also need to retrieve the additional clinical information about the patients – although it is not required by omnideconv. This is available in the Supplementary Table 1, included with the paper supplementary materials

2. Library loading

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3. Single cell data processing

Although not strictly required by omnideconv, we suggest performing quality control and filtering of the input scRNA-seq data according to the best practice (Heumos et al. 2023), to ensure the best training conditions for deconvolution algorithms. We first pre-process the single-cell dataset to remove low-quality cells. We will use the R package Seurat (Hao et al. 2023), which allows fast and easy manipulation of single-cell data. We will create a Seurat object with the cell counts and their metadata of interest curated by the authors, which include cell-type annotation on three levels of resolution:

single.cell.data <- Seurat::ReadMtx(
  mtx = 'C:/Users/c1041161/book_chapter/Wu_etal_2021_BRCA_scRNASeq/count_matrix_sparse.mtx',
  cells = 'C:/Users/c1041161/book_chapter/Wu_etal_2021_BRCA_scRNASeq/count_matrix_barcodes.tsv',
  features = 'C:/Users/c1041161/book_chapter/Wu_etal_2021_BRCA_scRNASeq/count_matrix_genes.tsv',
  feature.column = 1
)
single.cell.metadata <- read.table('C:/Users/c1041161/book_chapter/Wu_etal_2021_BRCA_scRNASeq/metadata.csv', 
                                   sep = ',',
                                   header = TRUE, 
                                   row.names = 1)

single.cell.data = CreateSeuratObject(single.cell.data, 
                                      project='Wu_dataset', 
                                      assay='RNA', 
                                      min.cells = 0, 
                                      min.features = 1, meta.data = single.cell.metadata)

We can have an overview of the number of cells per cell type in the dataset:

Number of cells per cell type
celltype_major number_cells
B-cells 3206
CAFs 6573
Cancer Epithelial 24489
Endothelial 7605
Myeloid 9675
Normal Epithelial 4355
Plasmablasts 3524
PVL 5423
T-cells 35214

In order to remove low quality cells, we will follow the best practices for single cell normalization (Heumos et al. 2023). We will perform quality control on each cell by considering metrics such as the number of total counts, the number of expressed features (genes), and the fraction of mitochondrial genes per cell. We will remove cells that have MAD=median(|Ximedian(X)|)MAD = median(|X_i - median(X)|), where

  • XiX_i is the respective metric of an observation ii (cell), for example the number of detected genes
  • MADMAD is the Mead Absolute Deviation, computed as MAD=median(|Ximedian(X)|)MAD = median(|X_i - median(X)|)
  • nn is set to 5 for the number of counts and expressed features, and to 3 for the fraction of mitochondrial genes

In order to do this filtering, we will create a function to identify the outliers for each metric

is_outlier <- function(SeuratObject, metric, nmads){
  eval(parse(text = paste0("M <- SeuratObject$",metric)))
  outlier <- (M < median(M) - nmads * mad(M)) | (M > median(M) + nmads * mad(M))
  return(outlier)
}

check_outliers_nFeature <- is_outlier(single.cell.data, 'nFeature_RNA', 5)
check_outliers_nCount <- is_outlier(single.cell.data, 'nCount_RNA', 5)
check_outliers_mito <- is_outlier(single.cell.data, 'percent.mito', 3)

non_outliers_nFeature <- names(check_outliers_nFeature[!check_outliers_nFeature])
non_outliers_nCount <- names(check_outliers_nCount[!check_outliers_nCount])
non_outliers_mito <- names(check_outliers_mito[!check_outliers_mito])

We will retain only those that satisfy all three of the conditions described above.

non_outliers <- intersect(non_outliers_nFeature, non_outliers_nCount) %>% 
  intersect(non_outliers_mito)

single.cell.data <- subset(single.cell.data, cells = non_outliers)

as.data.frame(table(single.cell.data$celltype_major, dnn = list("celltype_major")), responseName = "number_cells")
#>      celltype_major number_cells
#> 1           B-cells         3150
#> 2              CAFs         6154
#> 3 Cancer Epithelial        16613
#> 4       Endothelial         6991
#> 5           Myeloid         8855
#> 6 Normal Epithelial         3533
#> 7      Plasmablasts         3164
#> 8               PVL         5120
#> 9           T-cells        34991
Number of cells per cell type after quality filtering
celltype_major number_cells
B-cells 3150
CAFs 6154
Cancer Epithelial 16613
Endothelial 6991
Myeloid 8855
Normal Epithelial 3533
Plasmablasts 3164
PVL 5120
T-cells 34991

4. Bulk data preprocessing

We will now read in the bulk sequencing data file, which consists of 24 samples.

bulk.data <- read.table('C:/Users/c1041161/book_chapter/GSE176078_Wu_etal_2021_bulkRNAseq_raw_counts.txt', skip=1)

header <- read.table('C:/Users/c1041161/book_chapter/GSE176078_Wu_etal_2021_bulkRNAseq_raw_counts.txt', 
                     header = FALSE, nrows = 1, skipNul = TRUE, sep='\t')

colnames(bulk.data) <- c('Genes', gsub('A|N', '', header[2:25]))

bulk.data <- bulk.data[bulk.data$Genes != '', ]
bulk.data <- column_to_rownames(bulk.data, 'Genes')
bulk.data <- as.matrix(bulk.data)

5. Subsampling of single cell data

The various methods included in omnideconv rely on the single cell dataset that will be used to train them for the deconvolution of those specific cell types. This training involves the optimization of internal features of the methods and can happen in different ways. Some methods use the single cell data to build a ‘signature matrix’, i.e. a reduced transcriptional fingerprints of the cell types provided, while others use this data in a statistical or deep learning model. Since single cell datasets can often encompass thousands of cells, we will need to subsample it in order to be able to run the analysis on our machines. In this case we will retain a maximum of 200 cells per cell type, but this step can be costumed, or eventually skipped, depending on the computational resources available.

max_cells_per_celltype = 200

sampled.metadata <- single.cell.data@meta.data %>%
      rownames_to_column(., 'barcode') %>%
      group_by(., celltype_major) %>% 
      nest() %>%            
      mutate(n =  map_dbl(data, nrow)) %>%
      mutate(n = min(n, max_cells_per_celltype)) %>%
      ungroup() %>% 
      mutate(samp = map2(data, n, sample_n)) %>% 
      select(-data) %>%
      unnest(samp)

single.cell.data.sampled <- subset(single.cell.data, cells = sampled.metadata$barcode)


#as.data.frame(table(single.cell.data.sampled$celltype_major, dnn = list("celltype_major")), responseName = "number_cells")
Number of cells per cell type after subsampling
celltype_major number_cells
B-cells 200
CAFs 200
Cancer Epithelial 200
Endothelial 200
Myeloid 200
Normal Epithelial 200
Plasmablasts 200
PVL 200
T-cells 200

6. Deconvolution of the bulk data

Each methods has different requirements, but in general to compute the deconvolution results we will need the single cell counts matrix, the cell type annotations and the information on the individual/experiment fom which the cells were retrieved (batch ID).

counts.matrix <- as.matrix(single.cell.data.sampled@assays$RNA@counts)
cell.type.annotations <- single.cell.data.sampled$celltype_major
batch.ids <- single.cell.data.sampled$orig.ident

6.1 Deconvolution with DWLS

Now we’re going to deconvolute the bulk dataset with different methods. The first one we are going to use is called DWLS (Tsoucas et al. 2019) and performs the deconvolution in a two-steps process. First, the single cell data is used to build a signature matrix using the omnideconv function build_model. DWLS looks for differentially expressed genes that discriminate across cell types, and can do so with two approaches based either on the Seurat (Hao et al. 2023) “bimod” test (McDavid et al. 2012) or on MAST (Finak et al. 2015). MAST improves the former model, but has an increased computational requirement (Nault et al. 2022). The authors recommend using this method on the smaller datasets, and to switch to Seurat if the analysis with MAST cannot be completed. To reduce MAST’s computational time, we introduced a second version of the MAST-based function (mast_optimized) that speeds up the process compared to the original implementation:

# We need to insert the normalization as well
signature.matrix.dwls <- omnideconv::build_model(single_cell_object = counts.matrix,
                        cell_type_annotations = cell.type.annotations,
                        method = 'dwls', 
                        dwls_method = 'mast_optimized')

This signature is optimized so that the genes selected are the ones that help to discriminate across cell types.

The signature is now used for the deconvolution of the bulk RNAseq, which is performed with the omnideconv function deconvolute. DWLS computes the cell fractions performing one of Ordinary Least Squares (OLS) Regression, Support Vector Regression (SVR) or the Dampened Weighted Least Squares Regression (DWLS) that was introduced with the method. This last regression method is shown to outperform the others when it comes to the detection of rare cell types:

deconvolution.results.dwls <- deconvolute(bulk_gene_expression = bulk.data,
                                          signature = signature.matrix.dwls,
                                          method='dwls',
                                          dwls_submethod = 'DampenedWLS')

We will now obtain, for every sample, a set of cell type fractions for each cell type that was included in the provided single cell dataset.

Cell type fractions obtained by DWLS
B-cells CAFs Cancer Epithelial Endothelial Myeloid Normal Epithelial Plasmablasts PVL T-cells
CID3586 0.231 0.238 0.170 0.083 0.005 0.035 0.000 0.022 0.215
CID3921 0.026 0.177 0.145 0.114 0.019 0.068 0.004 0.000 0.447
CID3941 0.000 0.267 0.373 0.091 0.002 0.180 0.000 0.030 0.059
CID3948 0.466 0.107 0.264 0.049 0.007 0.000 0.001 0.020 0.086
CID3963 0.112 0.208 0.000 0.088 0.103 0.345 0.000 0.020 0.123
CID4066 0.049 0.342 0.174 0.102 0.019 0.155 0.000 0.049 0.111
CID4067 0.000 0.338 0.477 0.084 0.007 0.018 0.000 0.006 0.071
CID4290 0.000 0.408 0.417 0.068 0.072 0.005 0.000 0.024 0.006
CID4398 0.000 0.220 0.143 0.119 0.006 0.204 0.000 0.034 0.275
CID44041 0.332 0.161 0.000 0.122 0.006 0.264 0.000 0.032 0.084
CID4461 0.319 0.138 0.292 0.084 0.028 0.000 0.003 0.020 0.116
CID4463 0.033 0.123 0.560 0.069 0.067 0.036 0.000 0.039 0.074
CID4465 0.226 0.287 0.066 0.099 0.070 0.206 0.004 0.013 0.029
CID4471 0.009 0.288 0.000 0.182 0.046 0.296 0.000 0.143 0.035
CID4495 0.366 0.066 0.023 0.071 0.066 0.274 0.002 0.005 0.126
CID44971 0.286 0.099 0.000 0.083 0.002 0.300 0.000 0.010 0.219
CID4513 0.001 0.398 0.000 0.143 0.397 0.000 0.001 0.056 0.005
CID4515 0.456 0.185 0.000 0.092 0.050 0.131 0.002 0.000 0.085
CID4523 0.000 0.219 0.381 0.165 0.025 0.166 0.000 0.000 0.044
CID4530 0.085 0.317 0.284 0.083 0.034 0.068 0.000 0.040 0.088
CID4535 0.075 0.062 0.681 0.049 0.007 0.000 0.000 0.029 0.097
CID4040 0.305 0.217 0.272 0.052 0.030 0.000 0.002 0.009 0.114
CID3838 0.169 0.200 0.332 0.085 0.058 0.077 0.000 0.027 0.052
CID3946 0.389 0.147 0.000 0.081 0.038 0.256 0.000 0.004 0.085

We can also visualise the results as a barplot trough the built-in plot_deconvolution function

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6.2 Deconvolution with BayesPrism

The third method we will use is BayesPrism (Chu et al. 2022). This method is based on a Bayesian framework and models the transcriptomic expression observed in the scRNA-seq dataset. It then uses this information to dissect te bulk RNA-seq.

# BayesPrism deconvolution

deconvolution.results.bayesprism <- deconvolute(bulk_gene_expression = bulk.data,
                                           single_cell_object = counts.matrix,
                                           cell_type_annotations = cell.type.annotations,
                                           signature=NULL,
                                           method = 'bayesprism', 
                                           n_cores=12)

We can visualize the results as before:

omnideconv::plot_deconvolution(list('bayesprism' = deconvolution.results.bayesprism), "bar", "method", "Spectral")
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7. Deconvolution of the bulk data at a lower resolution

The considered single-cell breast cancer dataset includes cell-type annotations at three levels of resolution: celltype_major, celltype_minor, and celltype_subset, which distinguish 9, 29, and 58 cell types respectively. The different cell-type annotations can be accessed with:

single.cell.data$celltype_major      # Major annotation
single.cell.data$celltype_minor      # Minor annotation
single.cell.data$celltype_subset     # Subset annotation

These additional annotations provide a cell-type classification at a finer resolution. For instance, at the celltype_major level, we only have the T cell population, while at the celltype_minor level, we can distinguish between CD4+ and CD8+ T cells. In the following, we will again perform deconvolution analysis with DWLS but, this time, using the celltype_minor information. We will subsample the dataset as before, this time considering the second level of resolution for the cell types, and extract the objects needed for deconvolution.

max_cells_per_celltype = 200


sampled.metadata <- single.cell.data@meta.data %>%
      rownames_to_column(., 'barcode') %>%
      group_by(., celltype_minor) %>% 
      nest() %>%            
      mutate(n =  map_dbl(data, nrow)) %>%
      mutate(n = min(n, max_cells_per_celltype)) %>%
      ungroup() %>% 
      mutate(samp = map2(data, n, sample_n)) %>% 
      select(-data) %>%
      unnest(samp)

single.cell.data.sampled <- subset(single.cell.data, cells = sampled.metadata$barcode)
Number of cells per cell type (minor resolution) after subsampling
celltype_minor number_cells
B cells Memory 200
B cells Naive 200
CAFs MSC iCAF-like 200
CAFs myCAF-like 200
Cancer Basal SC 200
Cancer Cycling 200
Cancer Her2 SC 200
Cancer LumA SC 200
Cancer LumB SC 200
Cycling PVL 37
Cycling T-cells 200
Cycling_Myeloid 200
DCs 200
Endothelial ACKR1 200
Endothelial CXCL12 200
Endothelial Lymphatic LYVE1 183
Endothelial RGS5 200
Luminal Progenitors 200
Macrophage 200
Mature Luminal 200
Monocyte 200
Myoepithelial 200
NK cells 200
NKT cells 200
Plasmablasts 200
PVL Differentiated 200
PVL Immature 200
T cells CD4+ 200
T cells CD8+ 200
counts.matrix <- as.matrix(single.cell.data@assays$RNA@counts)
cell.type.annotations <- single.cell.data$celltype_minor
batch.ids <- single.cell.data$orig.ident

signature.matrix.dwls.minor <- omnideconv::build_model(single_cell_object = counts.matrix,
                        cell_type_annotations = cell.type.annotations,
                        method = 'dwls', 
                        dwls_method = 'mast_optimized')

deconvolution.results.dwls.minor <- deconvolute(bulk_gene_expression = bulk.data,
                                          signature = signature.matrix.dwls.minor,
                                          method='dwls',
                                          dwls_submethod = 'DampenedWLS')

We can visualize the results as before:

omnideconv::plot_deconvolution(list('dwls' = deconvolution.results.dwls.minor), "bar", "method", "Spectral")
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8. Comparison of cell fractions across conditions

We can consider as well the metadata provided with the original paper, which include patient’s data, cancer subtype information and treatment details. We can first harmonize the sample names, and then combine all this information with the deconvolution results in one dataframe.

patient.metadata <- read_excel("C:/Users/c1041161/book_chapter/41588_2021_911_MOESM4_ESM.xlsx", sheet = 1, skip = 3) %>%
  select(., c(1, 4, 5, 11, 12))
colnames(patient.metadata) <- c('Sample', 'Grade', 'Cancer_type', 'IHC_subtype', 'Treatment')

patient.metadata$Sample <- gsub('-', '', patient.metadata$Sample) %>%
  paste0('CID', .)

patients.results <- rownames_to_column(as.data.frame(deconvolution.results.dwls), 'Sample') %>%
  gather(., key='celltype', value='cell_fraction', -Sample) %>%
  left_join(., patient.metadata)

Each condition has a different number of samples:

We can visualize the estimated cell fractions across samples with a boxplot, and group the results either by IHC subtype or by treatment status

plot of chunk unnamed-chunk-27

HER2-positive samples are enriched in T cells, while the ER-positive and triple negative breast cancer (TNBC) samples show an enrichment in cancer and normal epithelial cells, respectively. The patient metadata includes information about eventual treatments undergone by the patients. We can see that 5 out of the 24 patients were treated with Neoadjuvant and/or Paclitaxel, while the other 19 were untreated. We can therefore visualize the distribution of cell fractions across treated and untreated patients

plot of chunk unnamed-chunk-28

The major differences in this case are the enrichment of the CAFs and normal epithelial cells in the treated samples, as opposed to the cancer epithelial cells and B cells which have a higher median cell fraction in the untreated samples.

As mentioned before, with the lower level of annotations we can identify additional cell types, such as CD4+/CD8+ subtypes, Fibroblasts and cancer cells. We can therefore visualize the estimated cell fractions in a boxplot, focusing in particular on the CD4+ and CD8+ T-cell subtypes.

plot of chunk unnamed-chunk-29

The estimates for the CD8+ T cells seem to be very low for all samples. On the other hand, in the HER2/ER positive samples the cell fractions estimated for the CD4+ T cells seem to be significantly higher than in the other subtypes. The estimates for the CD8+ T cells are close to zero for almost every sample, with very low fractions detected for the HER2+/ER+ ones.

Similarly, we can visualize the distribution of the estimates for the cancer associated fibroblasts (CAFs), respectively the myoblastic CAFs (myCAF) and the bone marrow derived inflammatory CAFs (MSC iCAF).

plot of chunk unnamed-chunk-30

Here we can notice that, while the myoblastic CAFs have a comparable median value, the MSC CAFs have higher infiltration in the samples characterized as HER2+/ER+ subtype.

Finally, we can visualize the distribution of the different cancer cells molecular subtypes that were described by the authors.

plot of chunk unnamed-chunk-31

We can see that the HER2+ shows an overrepresentation of the cancer cells described as HER2-like by the authors. Similarly, the ER+ samples show higher fractions of Luminal B cancer cells. These findings on bulk RNA-seq are concordant with the subtypings that the authors described on the single cell RNA-seq (‘Results - scSubtype’).

References

Chu, Tinyi, Zhong Wang, Dana Pe’er, and Charles G. Danko. 2022. “Cell Type and Gene Expression Deconvolution with Bayesprism Enables Bayesian Integrative Analysis Across Bulk and Single-Cell Rna Sequencing in Oncology.” Nature Cancer 3 (4): 505–17. https://doi.org/10.1038/s43018-022-00356-3.

Finak, Greg, Andrew McDavid, Masanao Yajima, Jingyuan Deng, Vivian Gersuk, Alex K. Shalek, Chloe K. Slichter, et al. 2015. “MAST: A Flexible Statistical Framework for Assessing Transcriptional Changes and Characterizing Heterogeneity in Single-Cell Rna Sequencing Data.” Genome Biology 16 (1). https://doi.org/10.1186/s13059-015-0844-5.

Hao, Yuhan, Tim Stuart, Madeline H. Kowalski, Saket Choudhary, Paul Hoffman, Austin Hartman, Avi Srivastava, et al. 2023. “Dictionary Learning for Integrative, Multimodal and Scalable Single-Cell Analysis.” Nature Biotechnology 42 (2): 293–304. https://doi.org/10.1038/s41587-023-01767-y.

Heumos, Lukas, Anna C. Schaar, Christopher Lance, Anastasia Litinetskaya, Felix Drost, Luke Zappia, Malte D. Lücken, et al. 2023. “Best Practices for Single-Cell Analysis Across Modalities.” Nature Reviews Genetics 24 (8): 550–72. https://doi.org/10.1038/s41576-023-00586-w.

McDavid, Andrew, Greg Finak, Pratip K. Chattopadyay, Maria Dominguez, Laurie Lamoreaux, Steven S. Ma, Mario Roederer, and Raphael Gottardo. 2012. “Data Exploration, Quality Control and Testing in Single-Cell qPCR-Based Gene Expression Experiments.” Bioinformatics 29 (4): 461–67. https://doi.org/10.1093/bioinformatics/bts714.

Nault, Rance, Satabdi Saha, Sudin Bhattacharya, Jack Dodson, Samiran Sinha, Tapabrata Maiti, and Tim Zacharewski. 2022. “Benchmarking of a Bayesian Single Cell Rnaseq Differential Gene Expression Test for Dose–Response Study Designs.” Nucleic Acids Research 50 (8): e48–e48. https://doi.org/10.1093/nar/gkac019.

Tsoucas, Daphne, Rui Dong, Haide Chen, Qian Zhu, Guoji Guo, and Guo-Cheng Yuan. 2019. “Accurate Estimation of Cell-Type Composition from Gene Expression Data.” Nature Communications 10 (1). https://doi.org/10.1038/s41467-019-10802-z.

Wu, Sunny Z., Ghamdan Al-Eryani, Daniel Lee Roden, Simon Junankar, Kate Harvey, Alma Andersson, Aatish Thennavan, et al. 2021. “A Single-Cell and Spatially Resolved Atlas of Human Breast Cancers.” Nature Genetics 53 (9): 1334–47. https://doi.org/10.1038/s41588-021-00911-1.