Author: Xudong Han and Xuejiang Guo
Date: 2023-08-08

# scapGNN: Graph Neural Network-Based Framework for Single Cell Active Pathways and Gene Modules Analysis

#### Xudong Han and Xuejiang Guo

#### 2023-08-08

    library(scapGNN)

# Introduction

With the widespread availability of single-cell sequencing technology, numerous single-cell RNA-seq and ATAC-seq data are collected into public databases. However, the single-cell data have characteristics such as drop-out events and low library sizes. How to leverage these data to parse cellular heterogeneity at the pathway level and identify activated gene modules with multiple cell phenotypes is still a challenge.

Here, we propose `scapGNN`, an accurate, effective, and robust framework to comprehensively parse single-cell data at the pathway level from single-omics data or multi-omics integration data. The `scapGNN` model takes single-cell gene expression profiles or the gene activity matrix of scATAC-seq as input data. As shown in Figure 1A, the computing architecture consists of three modules:

1. A depp neural network-based autoencoder, which is used to extract cellular features, gene features, and mine latent associations between genes and cells.

2. A graph convolutional network-based graph autoencoder, which is used to construct cell-cell association network and gene-gene association network.

3. Random walk with restart (RWR), which is used to calculate the activity score of a pathway or gene set in each cell.

For the SNARE-seq dataset, `scapGNN` can integrate gene-cell association networks from scATAC-seq and scRNA-seq into one combined network by the Brown’s extension of the Fisher’s combined probability test. According to the combined gene-cell association network, pathway activity scores or activated gene modules are supported by single-cell multi-omics (Figure 2). `scapGNN` also designs abundant visualization programs to clearly display the analysis results. Taken together, its capabilities enable `scapGNN` to calculate pathway activity scores in a single cell, assess pathway heterogeneity between cells, identify key active genes in pathway, mine gene modules under multiple cell phenotypes, and provide gene and cell network data (Figure 1B).

Figure 1 Overview of the scapGNN framework.

Figure 2 The workflow of integrating scRNA-seq data and scATAC-seq data by scapGNN.

# Data preprocessing

## single-cell RNA-seq data

Single-cell gene expression profiles data needs to pass `Seurat`’s pre-processing workflow. These represent the selection and filtration of cells based on QC metrics, data normalization, and the detection of highly variable features.

    library(Seurat)

    # Load the PBMC dataset (Case data for seurat)
    pbmc.data <- Read10X(data.dir = "../data/pbmc3k/filtered_gene_bc_matrices/hg19/")

    # Our recommended data filtering is that only genes expressed as non-zero in more than
    # 1% of cells, and cells expressed as non-zero in more than 1% of genes are kept.
    pbmc <- CreateSeuratObject(counts = pbmc.data, project = "case",
                                        min.cells = 3, min.features = 200)

    pbmc[["percent.mt"]] <- PercentageFeatureSet(pbmc, pattern = "^MT-")

    pbmc <- subset(pbmc, subset = nFeature_RNA > 200 & nFeature_RNA < 2500 & percent.mt < 5)

    # Normalizing the data.
    pbmc <- NormalizeData(pbmc, normalization.method = "LogNormalize")

    # Identification of highly variable features.
    pbmc <- FindVariableFeatures(pbmc, selection.method = 'vst', nfeatures = 2000)

    # Run Preprocessing() of scapGNN.
    Prep_data <- Preprocessing(pbmc,verbose=FALSE)

Detailed procedures and descriptions can be found in vignettes of `Seurat` (https://satijalab.org/seurat/).

    # Users can also directly input data in a data frame or matrix format which contains hypervariable genes and is log-transformed.
    data("Hv_exp")
    Prep_data <- Preprocessing(Hv_exp,verbose=FALSE)
    summary(Prep_data)
    #>               Length Class  Mode
    #> orig_dara      45000 -none- numeric
    #> cell_features  45000 -none- numeric
    #> gene_features  45000 -none- numeric
    #> ltmg_matrix        1 -none- function
    #> cell_adj        8100 -none- numeric
    #> gene_adj      250000 -none- numeric

## single-cell ATAC-seq data

First, the `Signac` package is used to quantify the activity of each gene in the genome by assessing the chromatin accessibility associated with each gene, and create a new gene activity matrix derived from the scATAC-seq data. For details, please refer to vignettes of `Signac` (https://satijalab.org/signac/index.html). As with single-cell gene expression profiles, the ensuing gene activity matrix is normalized and detected highly variable features by the `Seurat` package. Finally, the `Preprocessing()` of `scapGNN` further processes the data into the required format.

# Construct gene-cell association network

Using the `Preprocessing()` function results of scRNA-seq or scATAC-seq as input, the `ConNetGNN()` function evaluates whether gene-gene, cell-cell, and gene-cell are associated and levels of strength. Finally, an undirected and weighted gene-cell network is constructed and use for subsequent analysis.

The `ConNetGNN()` function is implemented based on `pytorch`, so an appropriate python environment is required:

- python >=3.9.7
- pytorch >=1.10.0 (CPU)
- sklearn >=0.0
- scipy >=1.7.3
- numpy >=1.19.5

We also provide environment files for conda: `/inst/extdata/scapGNN_env.yaml`. Users can install it with the command: `conda env create -f scapGNN_env.yaml`.

    library(coop)
    library(reticulate)
    library(parallel)
    # Data preprocessing
    data("Hv_exp")
    Prep_data <- Preprocessing(Hv_exp)

    # Run ConNetGNN()
    ConNetGNN_data <- ConNetGNN(Prep_data,python.path="../miniconda3/envs/scapGNN_env/python.exe")

    # View the content of the ConNetGNN() results.
    data(ConNetGNN_data)
    summary(ConNetGNN_data)
    #>                   Length Class  Mode
    #> cell_network        8100 -none- numeric
    #> gene_network      250000 -none- numeric
    #> gene_cell_network  45000 -none- numeric

For the SNARE-seq dataset, the `ConNetGNN()` results of scRNA-seq and scATAC-seq can be integrated into a combined gene-cell association network via `InteNet()`.

    library(ActivePathways)
    library(parallel)
    data(RNA_net)
    data(ATAC_net)
    RNA_ATAC_IntNet<-InteNet(RNA_net,ATAC_net,parallel.cores=1)

# Infer pathway activity score matrix at the single-cell level

For the gene-cell association network constructed by `ConNetGNN()`, the `scPathway()` function uses RWR algorithm to calculate the pathway activity score of each cell.

    library(parallel)
    library(utils)
    # Load the result of the ConNetGNN function.
    data(ConNetGNN_data)
    # We recommend the use of a compiler.
    # The compiler package can be used to speed up the operation.
    # library(compiler)
    # scPathway<- cmpfun(scPathway)
    scPathway_data<-scPathway(ConNetGNN_data,gmt.path=system.file("extdata", "KEGG_human.gmt", package = "scapGNN"),parallel.cores=1)

The result `scPathway_data` of `scPathway()` is a matrix of pathway activity scores, with rows as pathways and columns as cells.

    data(scPathway_data)
    scPathway_data[1:3,1:3]
    #>                                        H9_0h_1 H9_0h_2 H9_0h_3
    #> Ras signaling pathway [PATH:hsa04014]     0.20    0.19    0.17
    #> Rap1 signaling pathway [PATH:hsa04015]    0.08    0.10    0.10
    #> MAPK signaling pathway [PATH:hsa04010]    0.68    0.59    0.70

    library(pheatmap)
    pheatmap(scPathway_data)

Use the `plotGANetwork()` function to plot the gene network of pathways in the specified cell phenotype.

    library(igraph)
    #>
    #> Attaching package: 'igraph'
    #> The following objects are masked from 'package:stats':
    #>
    #>     decompose, spectrum
    #> The following object is masked from 'package:base':
    #>
    #>     union

    # Load data.
    data(ConNetGNN_data)
    data("Hv_exp")

    # Construct cell set.
    index<-grep("0h",colnames(Hv_exp))
    cellset<-colnames(Hv_exp)[index]

    # Construct gene set.
    pathways<-load_path_data(system.file("extdata", "KEGG_human.gmt", package = "scapGNN"))
    geneset<-pathways[[which(names(pathways)=="Tight junction [PATH:hsa04530]")]]

    plotGANetwork(ConNetGNN_data,cellset,geneset,vertex.label.dist=1.5,main = "Tight junction [PATH:hsa04530]")

The `plotGANetwork()` function can construct and display a cell community network.

    require(igraph)
    require(graphics)

    data(ConNetGNN_data)

    # Construct the cell phenotype vector.
    cell_id<-colnames(ConNetGNN_data[["cell_network"]])
    temp<-unlist(strsplit(cell_id,"_"))
    cell_phen<-temp[seq(2,length(temp)-1,by=3)]
    names(cell_id)<-cell_phen
    head(cell_id)
    #>        0h        0h        0h        0h        0h        0h
    #> "H9_0h_1" "H9_0h_2" "H9_0h_3" "H9_0h_4" "H9_0h_5" "H9_0h_6"
    plotCCNetwork(ConNetGNN_data,cell_id,edge.width=10)

# Identification of activated gene modules

The `cpGModule()` can identify cell phenotype activated gene module.

    require(parallel)
    #> Loading required package: parallel
    require(stats)

    # Load the result of the ConNetGNN function.
    data(ConNetGNN_data)
    data(Hv_exp)

    # Construct the cell set corresponding to 0h.
    index<-grep("0h",colnames(Hv_exp))
    cellset<-colnames(Hv_exp)[index]
    H9_0h_cpGM_data<-cpGModule(ConNetGNN_data,cellset,parallel.cores=1)
    #> Start RWR
    #> Start perturbation analysis
    summary(H9_0h_cpGM_data)
    #>     Genes                 AS               Pvalue               FDR
    #>  Length:30          Min.   :0.001181   Min.   :0.000e+00   Min.   :0.000e+00
    #>  Class :character   1st Qu.:0.001317   1st Qu.:3.630e-07   1st Qu.:2.018e-05
    #>  Mode  :character   Median :0.001513   Median :4.551e-05   Median :1.422e-03
    #>                     Mean   :0.001656   Mean   :4.345e-04   Mean   :8.280e-03
    #>                     3rd Qu.:0.001815   3rd Qu.:6.034e-04   3rd Qu.:1.325e-02
    #>                     Max.   :0.002608   Max.   :2.830e-03   Max.   :4.717e-02

Use the `plotGANetwork()` function to plot the gene network of module.

    library(igraph)

    # Load data.
    data(ConNetGNN_data)
    data("Hv_exp")
    data("H9_0h_cpGM_data")

    # Construct cell set.
    index<-grep("0h",colnames(Hv_exp))
    cellset<-colnames(Hv_exp)[index]

    # Construct gene set.
    geneset<-H9_0h_cpGM_data$Genes

    plotGANetwork(ConNetGNN_data,cellset,geneset,vertex.label.dist=1.5,main = "Gene network of 0h cells activated gene module")

For multiple cellular phenotypes (e.g. classification, time stage, etc.), `cpGModule()` identifies the activated gene modules individually. The `plotMulPhenGM()` function then quantifies the activation strength of genes in different cell phenotypes and displays gene networks under multiple cell phenotypes.

    require(igraph)
    require(grDevices)
    # Load the result of the ConNetGNN function.
    data(ConNetGNN_data)
    # Obtain cpGModule results for each cell phenotype.
    data(H9_0h_cpGM_data)
    data(H9_24h_cpGM_data)
    data(H9_36h_cpGM_data)
    data.list<-list(H9_0h=H9_0h_cpGM_data,H9_24h=H9_24h_cpGM_data,H9_36h=H9_36h_cpGM_data)
    plotMulPhenGM(data.list,ConNetGNN_data,margin=-0.05)

    # Set plotgraph=F to get the network data.
    data_graph<- plotMulPhenGM(data.list,ConNetGNN_data,margin=-0.05,plotgraph=F)
    #Obtain the adjacency matrix of the network by the method of igraph package.
    Gene_adj_matrix <- as.matrix(as_adjacency_matrix( data[["graph"]], attr="weight"))
    # The value of Gene_adj_matrix is the strength of the gene-gene association.

# Data analysis and visualization combined with Seurat package

The single-cell pathway activity matrix can be docked with the `Seurat` package to perform non-linear dimensional reduction, identify differentially active pathways, draw heat map, violin plot, and more.

    library(dplyr)
    library(Seurat)
    library(patchwork)

    data(scPathway_data)
    data <- CreateSeuratObject(counts = scPathway_data, project = "Pathway")
    all.genes <- rownames(data)
    data <- ScaleData(data, features = all.genes,verbose = FALSE)
    data <- RunPCA(data, features =all.genes,verbose = FALSE)
    data <- FindNeighbors(data, verbose = FALSE)
    data <- FindClusters(data, resolution = 0.5,verbose = FALSE)
    data <- RunUMAP(data,dims = 1:15, verbose = FALSE)

    cell_phen<-unlist(strsplit(colnames(scPathway_data),"_"))
    cell_phen<-cell_phen[seq(2,length(cell_phen)-1,by=3)]

    data@active.ident<-as.factor(cell_phen)

    # Visualize non-linear dimensional reduction
    DimPlot(data, reduction = "umap")

    # Identify differentially active pathways
    diff.pathways <- FindAllMarkers(data,verbose = FALSE)

    # violin plot
    VlnPlot(data, features = "MAPK signaling pathway [PATH:hsa04010]")

    # heat map
    DoHeatmap(data, features = row.names(scPathway_data))