Introduction
The scSNViz script provides a comprehensive visualization of single cell-specific expressed SNVs (sceSNVs) by mapping them onto a dimensionally reduced representation of the data (e.g., UMAP, t-SNE, or PCA, depending on the selected technique). It generates a series of plots that represent the basic statistics and properties of the SNVs in the dataset. scSNViz integrates existing packages, including Seurat, Slingshot, scType, and CopyKat.
Input
The script requires two inputs:
- STAR solo output directory (-m) that contains features.tsv.gz, barcodes.tsv.gz, and matrix.mtx.gz OR a Seurat object (-r) that was saved as a .RDS
- SCReadCounts output file (-t) that is tab-delimited and either a .tsv or .txt
Additionally, lists of SNVs with cell-barcode information not processed through SCReadCounts can be submitted in similar format using the (-t) option.
See sample files for reference.
Options
-h, --help
Show this help message and exit
-r RDS-FILE, --rds-file=RDS-FILE
RDS file containing Seurat object.
-m COUNTSMATRIX-FILE, --countsmatrix-file=COUNTSMATRIX-FILE
folder containing STARsolo output folder name that contains
the following files:
barcodes.tsv.gz, features.tsv.gz, and matrix.mtx.gz
-t SNV-FILE, --snv-file=SNV-FILE
scReadCounts file
-w DIMENSIONALITY-REDUCTION, --dimensionality-reduction=DIMENSIONALITY-REDUCTION
options include tSNE, PCA, UMAP. Default=UMAP.
-x TH-VARS, --th-vars=TH-VARS
Threshold for number of sceSNVs. Default=0 (display cells with N_SNVs > 0).
-y TH-READS, --th-reads=TH-READS
Threshold for number of variant reads (N_VAR). Default=0 (consider as sceSNV positions covered with N_VAR > 0).
-c, --disable-title
Disable title for individual SNV plots. Default=F
-d, --disable-ind-plots
Disable individual SNV plots. Default=F.
-e, --disable-3d-axis
Disable axes in 3D plots. Default=F.
-g, --disable-slingshot
Disable slingshot curves in 3D plots. Default=F.
-i, --enable-sctype
Enable sctype to run. Default=F.
-j TISSUE-TYPE, --tissue-type=TISSUE-TYPE
tissue type for scType; options include:
Immunesystem, Pancreas, Liver, Eye, Kidney, Brain,
Lung, Adrenal, Heart, Intestine, Muscle, Placenta,
Spleen, Stomach, Thymus
-k COLOR-SCALE, --color-scale=COLOR-SCALE
if you would like to change the default color settings with
these options, you may use Blues, Reds, YlOrRd, YlGnBu, plasma, RdBu
-b, --enable-cell-border
Enable cell border. Default=F
-q, --enable-dynamic-cell-size
Enable cell size to depend on number of reads. Default=F
-s, --save-each-plot
Save plots as separate HTML files. Default=F
Output
Directory structure
scSNViz generates outputs for the set of the sceSNVs and for each individual sceSNV, as follows:
sample_SNVs_dimensionality_reduction_xry/
│
├── Exploratory_Combined_Plots.html
├── sample_SNVs-summary.txt
└── Figures_Individual_Plots_HTML/ (optional)
├── Cell_types_scType.html (optional)
├── CNVs_CopyKat.html (optional)
├── Median_VAF_RNA.html
├── Mean_VAF_RNA.html
├── N_REFreads.html
├── Total_VAF_RNA.html
├── N_sceSNVs.html
├── N_VARreads.html
├── Histogram_N_SNV.png
├── Histogram_N_VARreadsCounts.png
├── Histogram_MeanSNVsVAF.png
├── Histogram_TotalVAF.png
└── Individual_sceSNVs/
├── VARreads/
│ └── 3D VARreads plot HTML files for each sceSNV
├── REFreads/
│ └── 3D REFreads plot HTML files for each sceSNV
└── VAF/
└── 3D VAF plot HTML files for each sceSNV
Note: The Figures_Individual_Plots_HTML directory and its contents are optional and will only be generated if the user requests it using the -s option.
In sample_SNVs_dimensionality_reduction_xry, dimensionality_reduction indicates the choice of dimensionality reduction selected. x and y indicate the threshold for sceSNVs and the variant reads, respectively.
Description
For the set of the sceSNVs, the separately produced figures show the following:
MeanSNVsVAF: Histogram of mean VAF per SNV per cell
N_SNV: Histogram of the number of SNVs per cell
N_VARreadsCounts: Histogram of the number of Variant Reads per cell
TotalVAF: Histogram of the Total VAF per cell (VARreads/(VARreads + REFreads) per cell)
Cell_types_scType: 3D UMAP/t-SNE/PCA representation of the cell types identified by scType
CNVs_CopyKat.html: 3D UMAP/t-SNE/PCA representation of the copy number variations identified by CopyKat
Mean_VAF_RNA: 3D UMAP/t-SNE/PCA representation of mean VAF for each cell
Median_VAF_RNA: 3D UMAP/t-SNE/PCA representation of median VAF for each cell
N_sceSNVs: 3D UMAP/t-SNE/PCA representation of number of number of SNVs for each cell
N_VARreads: 3D UMAP/t-SNE/PCA representation of number of Variant Reads for each cell
N_REFreads: 3D UMAP/t-SNE/PCA representation of number of Reference Reads for each cell
Total_VAF_RNA: 3D UMAP/t-SNE/PCA representation of Total VAF per cell
sample_SNVs-summary: a text file of the summary statistics per cell
Individual_sceSNVs: contains 3D dmensionality reduction plots for individual sceSNV of the following:
- Expressed Variant Allele Fraction per cell (VAF_RNA)
- Number of Reference Reads per cell (REFreads)
- Number of Variant Reads per cell (VARreads)
Exploratory_Combined_Plots: displays all the separately generated plots above into one single html for modularity
Installation
Download the R file:
- scSNViz.r (Updated: 07/27/2024)
The following CRAN packages are required:
- optparse, stringr, openxlsx, HGNChelper, Seurat, ggplot2, dplyr, plotly, htmlwidgets, htmltools, jsonlite.
The following Bioconductor packages are required:
- glmGamPoi, slingshot.
Other packages:
- copykat
Note too that the matrixStats package (a dependancy of Seurat) needs to be downgraded to version 1.1.0:
> remotes::install_version("matrixStats", version="1.1.0")
Examples
% Rscript scSNViz.r -t sample_SNVs.txt -m SAMNXX_wasp_Solo.out/Gene/filtered/
% Rscript scSNViz.r -t sample_SNVs.txt -m SAMNXX_wasp_Solo.out/Gene/filtered/
--dimensionality-reduction=umap \
--th-vars=1 --th-reads=10 \
-i --tissue-type=Liver -c -d -u
% Rscript scSNViz.r -t sample_SNVs.txt -r sample_Seurat_object.rds \
--dimensionality-reduction=tsne \
--th-vars=1 --th-reads=10 \
-i --tissue-type=Immunesystem \
-c -d -e --color-scale=YlGnBu