single-cell mapper (scMappR)

Description

Single-cell mapper (scMappR) is an R package that allows users to better understand the cell-type contributions to a list of genes. This is completed by scaling the genes within a list to the likelihood that they’ll be expressed within each cell-type within a tissue. Furthermore, when a gene list is from a bulk RNA-seq, cell-type proportions can be estimated. In this instance, the summary statistics of identified differentially expressed genes are further scaled by the proportion of the cell-type in the population and normalized to the ratio of cell-type propotion in case/control. These functionalities require a signature matrix; or a gene by cell-type matrix filled with the likelihood a gene is found in each cell-type. scMappR provides pre-computed signature matrices from 330 published scRNA-seq datasets across ~150 tissues as well as the infrastructure to generate signature matrices from scRNA-seq count data. All of these functionalities can be applied using a handful of functions described below. For more details, please find out vignette at (links).

Installation

Currently, there is only a development version. scMappR relies on the following dependencies which should be downlaoded/updated with scMappR automatically. Please ensure that these packages are not open when installing scMappR.

• ggplot2 - CRAN
• pheatmap - CRAN
• graphics - CRAN
• Seurat - CRAN
• GSVA - Bioconductor
• stats - CRAN
• utils - CRAN
• pcaMethods - Bioconductor
• grDevices - CRAN
• gProfileR - CRAN
• limSolve - CRAN

Install GSVA and pcaMethods from bioconductor first, as devtools::install_github() will automatically install CRAN dependencies.

1. Github (Development Version)

{r install_developter, eval=FALSE}

if (!requireNamespace(“BiocManager”, quietly = TRUE)) install.packages(“BiocManager”) if (!requireNamespace(“devtools”, quietly = TRUE)) install.packages(“devtools”)

BiocManager::install(“pcaMethods”) BiocManager::install(“GSVA”)

devtools::install_github(“DustinSokolowski/scMappR”)



2. CRAN (Stable Release) -- Currently not available

{r install_cran, eval=FALSE}

if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")
if (!requireNamespace("devtools", quietly = TRUE))
install.packages("devtools")

BiocManager::install("pcaMethods")
BiocManager::install("GSVA")

install.packages("scMappR")


Link to data used in scMappR: https://github.com/DustinSokolowski/scMappR_Data

To run scMappR locally, please download all .rda files in this data download repository. In many of the functions, the “rda_path” argument can be changed to wherever you would like to download these files to. It however assumes “~/scMappR/data”.

Primary Functionalities of scMappR.

Below describes the primary ways scMappR can contextualize gene lists and process data. It is strongly reccomended to set toSave = TRUE in functions and, when appropraite, internet = TRUE. Otherwise scMappR will not print files/directories and many of the results will not be printed.

• tissue_scMappR_custom(): This function visualizes signature matrix, clusters subsetted genes, completes enrichment of individual cell-types and co-enrichment.
• tissue_scMappR_internal(): This function loops through every signature matrix in a particular tissue and generates heatmaps, cell-type preferences, and co-enrichment.
• tissue_by_celltype_enrichment(): This function completes a fishers exact test of an input list of genes against one of the two curated tissue by cell-type marker datasets from scMappR.
• scMappR_and_pathway_analysis(): This function generates scMappR Transformed Variables (STVs), visualies them in a heatmap, and completes pathway enrichment of STVs and bulk gene list.
• process_dgTMatrix_lists(): This function takes a list of count matrices, processes them, calls cell-types, and genreates signature matrices.

Cell-type markers in a list of genes.

Internal signature matrix.

Input a list of human or mouse gene symbols as well as a tissue of interest to intentify and visualize enriched cell-types and cell-types co-enriched by the same genes. This process is repeated for every signature matrix (i.e. scRNA-seq study) present for the inputted tissue.

{r tissue_scMappR_internal, eval=FALSE }

data(Preoptic_Area) Signature <- POA_example$$POA_Rank_signature rowname <- get_gene_symbol(Signature) rownames(Signature) <- rowname$$rowname genes <- rownames(Signature)[1:60] rda_path1 = “~/Documents/scMappR/data” internal <- tissue_scMappR_internal(genes,“mouse”,output_directory = “scMappR_Test”, tissue = “hypothalamus”,rda_path = rda_path1, toSave = TRUE)


#### Provided signature matrix.

Complete the same process but with a signature matrix and gene list provided by the user. Here, gene symbols do not have to be human or mouse, the symbols in the list must match the signature.

{r tissue_scMappR_custom, eval=FALSE}

data(POA_example)
Signature <- POA_example$POA_Rank_signature rowname <- get_gene_symbol(Signature) rownames(Signature) <- rowname$rowname
genes <- rownames(Signature)[1:200]

internal <- tissue_scMappR_custom(genes,Signature,output_directory = "scMappR_Test_custom", toSave = TRUE)


Non tissue-specific

While computing custom signature matrices, there are cell-type markers across tissues and studies. These markers are stored in a gmt file format and can be used for cell-type enrichment. This may be useful if there is not a particular tissue in mind. This function inputs a list of human or mouse gene symbols.

{r tissue_by_celltype_enrichment, eval=FALSE }

data(POA_example) POA_generes <- POA_example$$POA_generes POA_OR_signature <- POA_example$$POA_OR_signature POA_Rank_signature <- POA_example$$POA_Rank_signature Signature <- POA_Rank_signature rowname <- get_gene_symbol(Signature) rownames(Signature) <- rowname$$rowname genes <- rownames(Signature)[1:100]

enriched <- tissue_by_celltype_enrichment(gene_list = genes, species = “mouse”,p_thresh = 0.05, isect_size = 3)



### Scaling and visualizing Differentially Expressed Genes from bulk RNA-seq data

This function requires a normalized count matrix from RNA-seq, a signature matrix such that there are fewer cell-types than there are samples, and a list of differentially expressed genes (with log2Fold-Change and adjusted P-value).

Then, it will calculate scMappR Transformed Values (STVs) before estimating if there are changes in cell-type proportion between samples. If toSave = TRUE then STV's will be visualized. Additionally, if internet = TRUE, scMappR will iteratively re-order DEGs based on their STV's and complete pathway analysis with g:ProfileR or gprofiler2.

{r scMappR_and_pathway_analysis, eval=FALSE}

data(PBMC_scMappR)
bulk_DE_cors <- PBMC_example$bulk_DE_cors bulk_normalized <- PBMC_example$bulk_normalized
odds_ratio_in <- PBMC_example\$odds_ratio_in
case_grep <- "_female"
control_grep <- "_male"
max_proportion_change <- 10
theSpecies <- "human"

toOut <- scMappR_and_pathway_analysis(bulk_normalized, odds_ratio_in,
bulk_DE_cors, case_grep = case_grep,
control_grep = control_grep, rda_path = "",
max_proportion_change = 10, print_plots = TRUE,
plot_names = "scMappR_vignette_", theSpecies = "human",
output_directory = "scMappR_vignette_",
sig_matrix_size = 3000, up_and_downregulated = TRUE,
internet = TRUE, toSave = TRUE)


Generating a signature matrix and processing scRNA-seq count data

A matrix, dgTMatrix, or list of these matrices are inputted where the rows are genes and the columns are indiviudal cells. The gene names must be human or mouse gene symbols. If the dataset is being processed from PanglaoDB, then the rownames are GeneSymbol-ENSMBL, here, set species_name = -9.

This function returns the signature matrix and cell-type labels. If toSave = TRUE signature matrices, all cell-type markers, the average expression of genes from each cell-type, and cell-type labels from gsva are stored as files in the working directory. Additionally, if saveSCObject = TRUE, then the Seurat object is also saved in the working directory.

{r process_dgTMatrix_lists, eval=FALSE}

data(sm) toProcess <- list(example = sm) tst1 <- process_dgTMatrix_lists(toProcess, name = “testPropcess”, species_name = -9, naming_preference = “eye”, rda_path = “~/scMappR/data”, panglao_set = “TRUE”, toSave = TRUE, saveSCObject = TRUE)