Outputs of each step in the scRNAbox pipeline

Introduction

The outputs of each Step of the scRNAbox pipeline are deposited into a step-specific folder in the working directory which contains three sub folders:

working_directory
└──step1
    ├── figs1
    ├── info1
    └── objs1
  • The figs/ folder contains figures;
  • The info/ folder contains text files and tables;
  • The objs/ folder contains intermediate Seurat RDS objects.

Note: If users re-run an Analytical Step, the outputs from the previous run will automatically be overwritten. If you do not want to lose the outputs from a previous run, it is important to copy the materials to a separate directory. One exception to this is when annotating data in Step 7; users can re-run the annotate step as many times as they wish and each interation will add a new metadata column to the already existing Seurat object.

Outputs

Step 1: FASTQ to gene expression matrix

All of the outputs of the CellRanger counts pipeline are produced. For more information on the outputs, please visit the CellRanger documentation.

Step 2: Create Seurat object and remove ambient RNA

Output type Name Description
Figure ambient_RNA_estimation_sample_name.pdf Sample-specific probability density plot showing the ambient RNA estimation. For more information see here
Figure ambient_RNA_markers_sample_name.pdf Sample-specific figure showing the marker genes used for ambient RNA estimation. For more information see [here]https://cran.r-project.org/web/packages/SoupX/vignettes/pbmcTutorial.html)
Figure vioplot_sample_name.pdf Sample-specific violin plot showing the distribution of cells according to QC metrics
Figure zoomed_in_vioplot_sample_name.pdf Sample-specific violin plot showing the distribution of cells according to QC metrics. The minimum value to the mean is shown.
Figure cell_cycle_dim_plot_sample_name.pdf Sample-specific principal component analysis of cell-cycle genes, colour-coded by the cell cycle score of each cell.
Info sample_name_ambient_rna_summary.rds Sample-specific summary of ambient RNA estimation by SoupX
Info sample_name_RNA.txt Sample-specific sparse matrix of RNA assay
Info estimated_ambient_RNA_sample_name.txt Sample-specific ambient RNA estimation.
Info MetaData_sample_name.txt Sample-specific dataframe showing the Seurat object metadata
Info meta_info_sample_name.txt Sample-specific text file showing the column names of the Seurat object metadata
Info summary_sample_name.txt Sample-specific text file showing the summary of QC metrics (Minimum, 1st Quartile, Median, Mean, 3rd Quartile, Maximum)
Info sessionInfo.txt Session information for the R session
Data object sample_name.rds Sample-specific intermediate Seurat RDS object

Step 3: Quality control and generation of filtered data objects

Output type Name Description
Figure dimplot_pca_sample_name.pdf Sample-specific PCA showing the first two PCs
Figure elbow_sample_name.pdf Elbow plot to visualize the percentage of variance explained by each PC
Figure filtered_QC_vioplot_sample_name.pdf Sample-specific violin plot showing the distribution of cells according to QC metrics after filtering
Figure VariableFeaturePlot_sample_name.pdf Sample-specific figure showing the most variably expressed genes
Info sample_name_RNA.txt Sample-specific sparse matrix of RNA assay
Info MetaData_sample_name.txt Sample-specific dataframe showing the Seurat object metadata
Info meta_info_sample_name.txt Sample-specific text file showing the column names of the Seurat object metadata
Info most_variable_genes_sample_name.txt Sample-specific text file showing the column names of the Seurat object metadata
Info summary_sample_name.txt Sample-specific text file showing the summary of QC metrics (Minimum, 1st Quartile, Median, Mean, 3rd Quartile, Maximum)
Info sessionInfo.txt Session information for the R session
Data object sample_name.rds Sample-specific intermediate Seurat RDS object

Step 4: Doublet removal (standard track)

Output type Name Description
Figure sample_nameDF.classifications.pdf Sample-specific UMAP plot showing droplet classifications (singlet or doublet)
Figure sample_doublet_summary.pdf Sample-specific violin plot showing pANN value across singlet and doublet assignments; sample-specific bar plot showing the number of singlets and doublets.
Info n_predicted_doublets_sample_name.txt Sample-specific text file showing the number of identified doublets.
Info sample_name_RNA.txt Sample-specific sparse matrix of RNA assay
Info MetaData_sample_name.txt Sample-specific dataframe showing the Seurat object metadata
Info meta_info_sample_name.txt Sample-specific text file showing the column names of the Seurat object metadata
Info sessionInfo.txt Session information for the R session
Data object sample_name.rds Sample-specific intermediate Seurat RDS object

Step 4: Demultiplexing and doublet detection (HTO track)

Output type Name Description
Figure run_name_DotPlot_HTO_MSD.pdf Run-specific dot plot showing the enrichment of barcode-labels across cell assignments
Figure run_name_Heatmap_HTO_MSD.pdf Run-specific heatmap showing the enrichment of barcode-labels across cell assignments
Figure run_name_Ridgeplot_HTO_MSD.pdf Run-specific ridge plot showing the enrichment of barcode-labels across cell assignments
Figure run_name_HTO_dimplot_pca_.pdf Run-specific PCA of antibody assay
Figure run_name_HTO_dimplot_umap_.pdf Run-specific UMAP of antibody assay
Figure run_name_nCounts_RNA_MSD.pdf Run-specific violin plot showing the number of unque transcripts across cell assignments
Info run_name.rds_old_antibody_label_MULTIseqDemuxHTOcounts.csv Run-specific list of sample-specific barcode labels used in the experiment
Info run_name_MULTIseqDemuxHTOcounts.csv Run-specific number of cells assigned to each sample
Info run_namefiltered_MULTIseqDemuxHTOcounts.csv Run-specific number of cells assigned to each sample after removal of doublet and negative droplets
Info run_name_meta_info_.txt Run-specific text file showing the column names of the Seurat object metadata
Info run_name_MetaData.txt Run-specific dataframe showing the Seurat object metadata
Info run_name_RNA.txt Run-specific sparse matrix of RNA assay
Info sessionInfo.txt Session information for the R session
Data object run_name.rds Run-specific intermediate Seurat RDS object

Step 5: Creation of a single Seurat object from all samples

Output type Name Description
Figure intergrated_DimPlot_pca.pdf PCA showing the first two PCs of integrated assay, colour-coded by sample
Figure integrated_DimPlot_umap.pdf UMAP of integrated assay, colour-coded by sample
Figure integrated_elbow.pdf Elbow plot to visualize the percentage of variance explained by each PC for the integrated assay
Figure integrated_Jackstraw.pdf Jackstraw plot to visualize the distribution of p-values for each PC for the integrated assay
Figure merge_DimPlot_pca.pdf PCA showing the first two PCs of merged object, colour-coded by sample
Figure merge_DimPlot_umap.pdf UMAP of merged object, colour-coded by sample
Figure merge_elbow.pdf Elbow plot to visualize the percentage of variance explained by each PC for the merged object
Figure merge_Jackstraw.pdf Jackstraw plot to visualize the distribution of p-values for each PC for the merged object
Info seu_int_RNA.txt Sparse matrix of integrated assay
Info seu_int_MetaData.txt Dataframe showing the integrated object metadata
Info integrated_meta_info_seu_step5.csv Text file showing the column names of the integrated object metadata
Info seu_merge_RNA.txt Sparse matrix of merged data object
Info seu_merge_MetaData.txt Dataframe showing the merged object metadata
Info merge_meta_info_seu_step5.csv Text file showing the column names of the merged object metadata
Info sessionInfo.txt Session information for the R session
Data object seu_step5.rds Integrated intermediate Seurat RDS object

Step 6: Clustering

Output type Name Description
Figure clustree_int.pdf Clustree plot showing the stability across the user-defied clustering resolutions
Figure integrated_snn_res.pdf UMAP at the user defined clustering-resolution
Figure ARI.pdf Mean and standard deviation of the Adjusted Rand Index (ARI) between clustering pairs at a user-defined resolution
Info clustering_ARI.xlsx Excel file showing the mean and standard deviation of the ARI between clustering pairs at a user-defined resolution
Info seu_RNA.txt Sparse matrix of integrated assay
Info seu_MetaData.txt Dataframe showing the Seurat object metadata
Info meta_info.csv Text file showing the column names of the Seurat object metadata
Info sessionInfo.txt Session information for the R session
Data object seu_step6.rds Intermediate Seurat RDS object

Step 7: Cluster annotation

Cluster annotation method Output type Name Description
Tool 1: Cluster marker GSEA Figure heatmap.pdf Heatmap showing the expression of the top marker genes across cells, stratified by cluster
Tool 1: Cluster marker GSEA Figure plotenrich.pdf Barplot showing the 20 most enriched terms for a particular cluster and cell type library
Tool 2: Profile known markers Figure module_score_gene_set.pdf UMAP plot showing the module score across cells for user-defined gene sets
Tool 2: Profile known markers Figure select_feature_dot_plot.pdf Dotplot showing the expression of user-defined features at the cluster level
Tool 2: Profile known markers Figure select_feature_violin_plot.pdf Violin plot showing the expression of user-defined features at the cluster level
Tool 2: Profile known markers Figure select_feature_feature_plot.pdf UMAP plots showing the expression of user-defined features at the cell level
Tool 3: Reference-based annotations Figure UMAP_transferred_labels.pdf UMAP plots showing the cluster annotations from the reference Seurat object projected onto the query Seurat object
Annotate Figure clustering_name_cluster_annotation.pdf UMAP plot of the integrated assay showing the cluster annotation
Annotate Figure clustering_name_split_cluster_annotation.pdf UMAP plot of the integrated assay showing the cluster annotation, split by sample
General Info meta_info_seu_step7.txt Text file showing the column names of the Seurat object metadata
General Info sessionInfo_marker.txt Session information for the R session
Tool 1: Cluster marker GSEA Info cluster_just_genes.xlsx Excel file showing the marker genes for each cluster
Tool 1: Cluster marker GSEA Info cluster_whole.xlsx Excel file showing the marker genes and corresponding summary statistics for each cluster
Tool 1: Cluster marker GSEA Info ClusterMarkers.csv csv file showing the marker genes and corresponding summary statistics for each cluster
Tool 1: Cluster marker GSEA Info top_sel.csv csv file showing the top n marker genes for each cluster. The user defined n in the execution parameters
Tool 1: Cluster marker GSEA Info Er.genes.csv Enrichment terms and the corresponding statistics for a particular cluster and cell type library
Tool 1: Cluster marker GSEA Data object ClusterMarkers.rds RDS object containing the marker genes for each cluster
Tool 2: Module score Info geneset_by_cluster.csv Mean module score across clusters for each user-defined gene set
Tool 3: Reference-based annotations Info reference_predictions_summary.xlsx Number of cells from each cluster assigned a particular annotation based of the reference
General Data object seu_step7.rds Intermediate Seurat RDS object

Step 8: Differential gene expression

DGE method Output type Name Description
general Figure contrast_name.pdf Volcano plot of showing differentially expressed genes
Cell-based DGE Info contrast_name_DEG.csv Differentially exppresed genes identified for the user-defined contrast
Sample-based DGE Info Aggregated_expression_summary.csv Aggregated counts across user-defined sample groups
Sample-based DGE Info SampleBased_DGEsummarytable.csv Number of differentially expressed genes in the positive and negative direction for each user-defined contrast
Sample-based DGE Info DGE_contrast_name.csv Differentially exppresed genes identified for the user-defined contrast
General Info seu_RNA.txt Sparse matrix of integrated assay
General Info seu_MetaData.txt Dataframe showing the Seurat object metadata
General Info meta_info.csv Text file showing the column names of the Seurat object metadata
General Info sessionInfo.txt Session information for the R session
General Data object seu_step8.rds Intermediate Seurat RDS object