5 Integrated ST pipeline

• Load the R packages.

# sc libraries
library(Seurat)
library(phateR)
library(DoubletFinder)
library(monocle)
library(slingshot)
library(GSVA)
library(limma)
library(plyr)
library(dplyr)
library(org.Mm.eg.db)
library(org.Hs.eg.db)
library(CellChat)
library(velocyto.R)
library(SeuratWrappers)
library(stringr)
library(scran)
library(ggpubr)
library(viridis)
library(pheatmap)
library(parallel)
library(reticulate)
library(SCENIC)
library(feather)
library(AUCell)
library(RcisTarget)
library(Matrix)
library(foreach)
library(doParallel)
library(clusterProfiler)
library(GPTCelltype)
library(openai)
library(HematoMap)
# st libraries
library(RColorBrewer)
library(Rfast2)
library(SeuratDisk)
library(abcCellmap)
library(biomaRt)
library(copykat)
library(gelnet)
library(ggplot2)
library(parallelDist)
library(patchwork)
library(markdown)
# getpot
library(getopt)
library(tools)
# HemaScopeR
library(HemaScopeR)

5.1 For 10X Visium data

• Run the integrated 10X Visium pipeline.

st_10x_visium_pipeline(
        input.data.dir = 'path/to/data',
        output.dir = '.',
        sampleName = 'Hema_ST',

        # For Step1 Loading
        rds.file = FALSE,
        filename = "filtered_feature_bc_matrix.h5",
        assay = "Spatial",
        slice = "slice1",
        filter.matrix = TRUE,
        to.upper = FALSE,

        # For Step2 QC
        Step2_QC = TRUE,
        min.gene = 200,
        min.nUMI = 500,
        max.gene = Inf,
        max.nUMI = Inf,
        min.spot = 0,
        bool.remove.mito = FALSE,
        species = 'mouse', # 'human' or 'mosue'

        # For Step3 Clustering
        Step3_Clustering = TRUE,
        normalization.method = 'SCTransform',
        npcs = 50,
        pcs.used = 1:10,
        resolution = 0.8,

        # For Step4 Find DEGs
        Step4_Find_DEGs = TRUE,
        only.pos = TRUE,
        min.pct = 0.25,
        logfc.threshold = 0.25,
        test.use = 'wilcox',

        # For Step5 SVF
        Step5_SVFs = TRUE,
        selection.method = 'moransi',
        n.top.show = 10,
        n.col.show = 5,

        # For Step6 Interaction
        Step6_Interaction = TRUE,
        commot.signaling_type = 'Secreted Signaling',
        commot.database = 'CellChat',
        commot.min_cell_pct = 0.05,
        commot.dis_thr = 500,
        commot.n_permutations = 100,

        # For Step7 CNV analysis
        Step7_CNV = TRUE,
        copykat.genome = NULL,
        copykat.LOW.DR = 0.05,
        copykat.UP.DR = 0.1,
        copykat.win.size = 25,
        copykat.distance = "euclidean",
        copykat.n.cores = 1,

        # For Step8 Deconvolution
        Step8_Deconvolution = TRUE,
        cell2loc.sc.h5ad.dir = NULL,
        cell2loc.sc.max.epoch = 1000,
        cell2loc.st.max.epoch = 10000,
        cell2loc.use.gpu = TRUE,
        cell2loc.use.dataset = 'LymphNode',

        # For Step9 Cellcycle
        Step9_Cellcycle = TRUE,
        s.features = NULL,
        g2m.features = NULL,

        # For Step10 Nich
        Step10_Niche = TRUE,
        coexistence.method = 'correlation',
        Niche.cluster.n = 4,

        # settings
        pythonPath = 'path/to/python',
        verbose = FALSE,
        genReport = TRUE
)

5.2 For MERFISH data

• Run the integrated MERFISH pipeline.

st_MERFISH_pipeline(
        input.data.dir,
        output.dir,
        sampleName = 'Hema_MERFISH',
        fov = 'fov',
        tech = 'Vizgen',
        
        # For Step1 Loading
        rds.file = FALSE,
        assay = NULL,
        Vizgen.z = 3L, 
        Akoya.type = 'inform',
        
        # For Step2 QC
        Step2_QC = TRUE,
        min.gene = 20,
        min.nUMI = 50,
        max.gene = Inf,
        max.nUMI = Inf,
        min.spot = 0,
        bool.remove.mito = FALSE,
        species = 'mouse', # 'human' or 'mosue'
        
        # For Step3 Clustering
        Step3_Clustering = TRUE,
        normalization.method = 'SCTransform',
        npcs = 50,
        pcs.used = 1:10,
        resolution = 0.4,
        
        # For Step4 Find DEGs
        Step4_Find_DEGs = TRUE,
        only.pos = TRUE,
        min.pct = 0.25,
        logfc.threshold = 0.25,
        test.use = 'wilcox',
        
        # For Step5 SVF
        Step5_SVFs = TRUE,
        selection.method = 'moransi',
        n.top.show = 10,
        n.col.show = 5,
        
        # For Step6 Interaction
        Step6_Interaction = TRUE,
        h5ad_path = NULL,
        counts_path = NULL,
        coordinates_path = NULL,
        coordinates_index_col = 0,
        counts_transpose = TRUE,
        commot.signaling_type = 'Secreted Signaling',
        commot.database = 'CellChat',
        commot.min_cell_pct = 0.05,
        commot.dis_thr = 500,
        commot.n_permutations = 100,
        
        # For Step7 Cellcycle
        Step7_Cellcycle = TRUE,
        s.features = NULL,
        g2m.features = NULL,
        
        verbose = FALSE,
        pythonPath = NULL
)

5.3 For stereo-seq data

• Run the integrated stereo-seq pipeline.

st_stereo_pipeline(
        input.data.dir,
        output.dir,
        sampleName = 'Hema_stereo',

        # For Step1 Loading
        data_type = 'gem',
        sep = '\t',
        bin_type = 'bins',
        bin_size = 100,
        spot_diameter = 80,
        is_sparse = TRUE,
        gene_list = NULL,
        region = NULL,
        assay = 'Spatial',

        # For Step2 QC
        Step2_QC = TRUE,
        min.gene = 20,
        min.nUMI = 50,
        max.gene = Inf,
        max.nUMI = Inf,
        min.spot = 0,
        bool.remove.mito = FALSE,
        species = 'mouse', # 'human' or 'mosue'

        # For Step3 Clustering
        Step3_Clustering = TRUE,
        normalization.method = 'SCTransform',
        npcs = 50,
        pcs.used = 1:10,
        resolution = 0.1,
        max.n.cluster = 30,

        # For Step4 Find DEGs
        Step4_Find_DEGs = TRUE,
        only.pos = TRUE,
        min.pct = 0.25,
        logfc.threshold = 0.25,
        test.use = 'wilcox',

        # For Step5 SVF
        Step5_SVFs = TRUE,
        selection.method = 'moransi',
        n.top.show = 10,
        n.col.show = 5,

        # For Step6 Interaction
        Step6_Interaction = TRUE,
        h5ad_path = NULL,
        counts_path = NULL,
        coordinates_path = NULL,
        coordinates_index_col = 0,
        counts_transpose = TRUE,
        commot.signaling_type = 'Secreted Signaling',
        commot.database = 'CellChat',
        commot.min_cell_pct = 0.05,
        commot.dis_thr = 500,
        commot.n_permutations = 100,

        # For Step7 Cellcycle
        Step7_Cellcycle = TRUE,
        s.features = NULL,
        g2m.features = NULL,

        verbose = FALSE,
        pythonPath = NULL
)