changeset 1:2b09ca1c5e41 draft

"planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/scater commit 154318f74839a4481c7c68993c4fb745842c4cce"
author iuc
date Thu, 09 Sep 2021 12:24:39 +0000 (2021-09-09)
parents a30f4bfe8f01
children 99f912d5af9f
files README.md environment.yml macros.xml scater-create-qcmetric-ready-sce.R scater-manual-filter.R scater-normalize.R scater-pca-filter.R scater-plot-dist-scatter.R scater-plot-exprs-freq.R scater-plot-pca.R scater-plot-tsne.R scater-plot-tsne.xml test-data/scater_exprs_freq.pdf test-data/scater_filtered_normalised.loom test-data/scater_manual_filtered.loom test-data/scater_pca_filtered.loom test-data/scater_pca_plot.pdf test-data/scater_qcready.loom test-data/scater_reads_genes_dist.pdf test-data/scater_reads_genes_dist_log.pdf test-data/scater_tsne_plot.pdf
diffstat 21 files changed, 191 insertions(+), 294 deletions(-) [+]
line wrap: on
line diff
--- a/README.md	Tue Sep 03 14:30:21 2019 -0400
+++ b/README.md	Thu Sep 09 12:24:39 2021 +0000
@@ -1,20 +1,20 @@
 # Wrappers for Scater
 
-This code wraps a number of [scater](https://bioconductor.org/packages/release/bioc/html/scater.html) functions as Galaxy wrappers. Briefly, the `scater-create-qcmetric-ready-sce` tool takes a sample gene expression matrix (usually read-counts) and a cell annotation file, creates a [SingleCellExperiment](https://bioconductor.org/packages/release/bioc/html/SingleCellExperiment.html) object and runs scater's `calculateQCMetrics` function (using other supplied files such as ERCC's and mitochondrial gene features).
+This code wraps a number of [scater](https://bioconductor.org/packages/release/bioc/html/scater.html) and [scuttle](https://bioconductor.org/packages/3.13/bioc/html/scuttle.html) functions as Galaxy wrappers. Briefly, the `scater-create-qcmetric-ready-sce` tool takes a sample gene expression matrix (usually read-counts) and a cell annotation file, creates a [SingleCellExperiment](https://bioconductor.org/packages/release/bioc/html/SingleCellExperiment.html) object and runs scater's `calculateQCMetrics` function (using other supplied files such as ERCC's and mitochondrial gene features).
 Various filter scripts are provided, along with some plotting functions for QC.
 
 
 ## Typical workflow
 
 1. Read in data with `scater-create-qcmetric-ready-sce`.
-2. Visualise it.\
+2. Visualise it.
    Take a look at the distribution of library sizes, expressed features and mitochondrial genes with `scater-plot-dist-scatter`.
-   Then look at the distibution of genes across cells with `scater-plot-exprs-freq`.
+   
 3. Guided by the plots, filter the data with `scater-filter`.\
    You can either manually filter with user-defined parameters or use PCA to automatically removes outliers.
 4. Visualise data again to see how the filtering performed using `scater-plot-dist-scatter`.\
    Decide if you're happy with the data. If not, try increasing or decreasing the filtering parameters.
-5. Normalise data with `scater-normalize`.
+
 6. Investigate other confounding factors.\
    Plot the data (using PCA) and display various annotated properties of the cells using `scater-plot-pca`.
 
@@ -51,10 +51,6 @@
 
 ---
 
-`scater-plot-exprs-freq.R`
-Plots mean expression vs % of expressing cells and provides information as to the number of genes expressed in 50% and 25% of cells.
-
----
 
 `scater-pca-filter.R`
 Takes SingleCellExperiment object (from Loom file) and automatically removes outliers from data using PCA. Save the filtered SingleCellExperiment object in Loom format.
@@ -74,18 +70,18 @@
 
 ---
 
-`scater-normalize.R`
-Compute log-normalized expression values from count data in a SingleCellExperiment object, using the size factors stored in the object. Save the normalised SingleCellExperiment object in Loom format.
+`scater-plot-pca.R`
+PCA plot of a SingleCellExperiment object. The options `-c`, `-p`, and `-s` all refer to cell annotation features. These are the column headers of the `-c` option used in `scater-create-qcmetric-ready-sce.R`.
 
 ```
-./scater-normalize.R -i test-data/scater_manual_filtered.loom -o test-data/scater_man_filtered_normalised.loom
+./scater-plot-pca.R -i test-data/scater_qcready.loom -c Treatment -p Mutation_Status -o test-data/scater_pca_plot.pdf
 ```
 
 ---
 
-`scater-plot-pca.R`
-PCA plot of a normalised SingleCellExperiment object (produced with `scater-normalize.R`). The options `-c`, `-p`, and `-s` all refer to cell annotation features. These are the column headers of the `-c` option used in `scater-create-qcmetric-ready-sce.R`.
+`scater-plot-tsne.R`
+t-SNE plot of a SingleCellExperiment object. The options `-c`, `-p`, and `-s` all refer to cell annotation features. These are the column headers of the `-c` option used in `scater-create-qcmetric-ready-sce.R`.
 
 ```
-./scater-plot-pca.R -i test-data/scater_man_filtered_normalised.loom -c Treatment -p Mutation_Status -o test-data/scater_pca_plot.pdf
+./scater-plot-tsne.R -i test-data/scater_qcready.loom -c Treatment -p Mutation_Status -o test-data/scater_tsne_plot.pdf
 ```
--- a/environment.yml	Tue Sep 03 14:30:21 2019 -0400
+++ b/environment.yml	Thu Sep 09 12:24:39 2021 +0000
@@ -4,11 +4,11 @@
   - bioconda
   - defaults
 dependencies:
-  - bioconductor-loomexperiment=1.2.0
-  - bioconductor-scater=1.12.2
-  - r-ggpubr=0.2.2
-  - r-mvoutlier=2.0.9
-  - r-optparse=1.6.2
+  - bioconductor-loomexperiment=1.10.1
+  - bioconductor-scater=1.20.0
+  - r-ggpubr=0.4.0
+  - r-optparse=1.6.6
+  - r-robustbase=0.93_8
   - r-rtsne=0.15
-  - r-scales=1.0.0
-  - r-workflowscriptscommon=0.0.4
+  - r-scales=1.1.1
+  - r-workflowscriptscommon=0.0.7
--- a/macros.xml	Tue Sep 03 14:30:21 2019 -0400
+++ b/macros.xml	Thu Sep 09 12:24:39 2021 +0000
@@ -1,14 +1,20 @@
 <macros>
-    <token name="@TOOL_VERSION@">1.12.2</token>
+    <token name="@TOOL_VERSION@">1.20.0</token>
+    <token name="@PROFILE@">20.01</token>
     <xml name="requirements">
         <requirements>
             <requirement type="package" version="@TOOL_VERSION@">bioconductor-scater</requirement>
-            <requirement type="package" version="1.6.2">r-optparse</requirement>
-            <requirement type="package" version="0.0.4">r-workflowscriptscommon</requirement>
-            <requirement type="package" version="1.2.0">bioconductor-loomexperiment</requirement>
+            <requirement type="package" version="1.6.6">r-optparse</requirement>
+            <requirement type="package" version="0.0.7">r-workflowscriptscommon</requirement>
+            <requirement type="package" version="1.10.1">bioconductor-loomexperiment</requirement>
             <yield />
         </requirements>
     </xml>
+    <xml name="bio_tools">
+        <xrefs>
+            <xref type="bio.tools">scater</xref>
+        </xrefs>
+    </xml>
     <xml name="citations">
         <citations>
             <citation type="doi">10.1093/bioinformatics/btw777</citation>
--- a/scater-create-qcmetric-ready-sce.R	Tue Sep 03 14:30:21 2019 -0400
+++ b/scater-create-qcmetric-ready-sce.R	Thu Sep 09 12:24:39 2021 +0000
@@ -8,67 +8,60 @@
 
 # parse options
 #SCE-specific options
-option_list = list(
+option_list <- list(
   make_option(
     c("-a", "--counts"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "A tab-delimited expression matrix. The first column of all files is assumed to be feature names and the first row is assumed to be sample names."
   ),
   make_option(
     c("-r", "--row-data"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Path to TSV (tab-delimited) format file describing the features. Row names from the expression matrix (-a), if present, become the row names of the SingleCellExperiment."
   ),
   make_option(
     c("-c", "--col-data"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Path to TSV format file describing the samples (annotation). The number of rows (samples) must equal the number of columns in the expression matrix."
   ),
   #The scater-specific options
   make_option(
-    c("--assay-name"),
-    action = "store",
-    default = 'counts',
-    type = 'character',
-    help= "String specifying the name of the 'assay' of the 'object' that should be used to define expression."
-  ),
-  make_option(
     c("-f", "--mt-controls"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Path to file containing a list of the mitochondrial control genes"
   ),
   make_option(
     c("-p", "--ercc-controls"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Path to file containing a list of the ERCC controls"
   ),
   make_option(
     c("-l", "--cell-controls"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Path to file (one cell per line) to be used to derive a vector of cell (sample) names used to identify cell controls (for example, blank wells or bulk controls)."
   ),
   make_option(
     c("-o", "--output-loom"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "File name in which to store the SingleCellExperiment object in Loom format."
   )
 )
 
-opt <- wsc_parse_args(option_list, mandatory = c('counts', 'output_loom'))
+opt <- wsc_parse_args(option_list, mandatory = c("counts", "output_loom"))
 
 # Read the expression matrix
 
@@ -79,64 +72,61 @@
 
 rowdata <- opt$row_data
 
-if ( ! is.null(opt$row_data) ){
+if (! is.null(opt$row_data)) {
   rowdata <- read.delim(opt$row_data)
 }
 
 coldata <- opt$col_data
 
-if ( ! is.null(opt$col_data) ){
+if (! is.null(opt$col_data)) {
   coldata <- read.delim(opt$col_data)
 }
 
 # Now build the object
-assays <- list(as.matrix(reads))
-names(assays) <- c(opt$assay_name)
-scle <- SingleCellLoomExperiment(assays = assays, colData = coldata, rowData = rowdata)
-# Define spikes (if supplied)
 
-
+sce <- SingleCellLoomExperiment(assays = list(counts = as.matrix(reads)), colData = coldata)
 #Scater options
 
 # Check feature_controls (only mitochondrial and ERCC used for now)
-feature_controls_list = list()
-if (! is.null(opt$mt_controls) && opt$mt_controls != 'NULL'){
-  if (! file.exists(opt$mt_controls)){
-    stop((paste('Supplied feature_controls file', opt$mt_controls, 'does not exist')))
+
+if (! is.null(opt$mt_controls)) {
+  if (! file.exists(opt$mt_controls)) {
+    stop((paste("Supplied feature_controls file", opt$mt_controls, "does not exist")))
   } else {
-    mt_controls <- readLines(opt$mt_controls)
-    feature_controls_list[["MT"]] <- mt_controls
+    mts <- readLines(opt$mt_controls)
   }
+} else {
+  mts <- NULL
 }
 
-if (! is.null(opt$ercc_controls) && opt$ercc_controls != 'NULL'){
-  if (! file.exists(opt$ercc_controls)){
-    stop((paste('Supplied feature_controls file', opt$ercc_controls, 'does not exist')))
+if (! is.null(opt$ercc_controls)) {
+  if (! file.exists(opt$ercc_controls)) {
+    stop((paste("Supplied feature_controls file", opt$ercc_controls, "does not exist")))
   } else {
     ercc_controls <- readLines(opt$ercc_controls)
-    feature_controls_list[["ERCC"]] <- ercc_controls
   }
 } else {
-  ercc_controls <- character()
+  ercc_controls <- NULL
 }
 
 # Check cell_controls
-cell_controls_list <- list()
-if (! is.null(opt$cell_controls) && opt$cell_controls != 'NULL'){
-  if (! file.exists(opt$cell_controls)){
-    stop((paste('Supplied feature_controls file', opt$cell_controls, 'does not exist')))
+
+if (! is.null(opt$cell_controls)) {
+  if (! file.exists(opt$cell_controls)) {
+    stop((paste("Supplied feature_controls file", opt$cell_controls, "does not exist")))
   } else {
     cell_controls <- readLines(opt$cell_controls)
-    cell_controls_list[["empty"]] <- cell_controls
   }
+} else {
+  cell_controls <- NULL
 }
 
+# calculate QCMs
 
-# calculate QCMs
-scle  <- calculateQCMetrics(scle, exprs_values = opt$assay_name, feature_controls = feature_controls_list, cell_controls = cell_controls_list)
+sce <- addPerCellQC(sce, subsets = list(Mito = mts, ERCC = ercc_controls, cell_controls = cell_controls))
 
 # Output to a Loom file
 if (file.exists(opt$output_loom)) {
   file.remove(opt$output_loom)
 }
-export(scle, opt$output_loom, format='loom')
+export(sce, opt$output_loom, format = "loom")
--- a/scater-manual-filter.R	Tue Sep 03 14:30:21 2019 -0400
+++ b/scater-manual-filter.R	Thu Sep 09 12:24:39 2021 +0000
@@ -8,92 +8,94 @@
 library(scater)
 
 # parse options
-option_list = list(
+option_list <- list(
   make_option(
     c("-i", "--input-loom"),
     action = "store",
     default = NA,
-    type = 'character',
-    help = "A SingleCellExperiment object file in Loom format."
+    type = "character",
+    help = "A SingleCellExperiment object file in Loom format"
+  ),
+  make_option(
+    c("-l", "--library-size"),
+    action = "store",
+    default = 0,
+    type = "numeric",
+    help = "Minimum library size (mapped reads) to filter cells on"
+  ),
+  make_option(
+    c("-m", "--percent-counts-MT"),
+    action = "store",
+    default = 100,
+    type = "numeric",
+    help = "Maximum % of mitochondrial genes expressed per cell. Cells that exceed this value will be filtered out"
+  ),
+  make_option(
+    c("-f", "--expressed-features"),
+    action = "store",
+    default = 100,
+    type = "numeric",
+    help = "Remove cells that have less than the given number of expressed features"
   ),
   make_option(
     c("-d", "--detection-limit"),
     action = "store",
     default = 0,
-    type = 'numeric',
-    help = "Numeric scalar providing the value above which observations are deemed to be expressed"
-  ),
-  make_option(
-    c("-l", "--library-size"),
-    action = "store",
-    default = 0,
-    type = 'numeric',
-    help = "Minimum library size (mapped reads) to filter cells on"
+    type = "numeric",
+    help = "Number of reads mapped to a feature above which it to be deemed as expressed"
   ),
   make_option(
-    c("-e", "--expressed-genes"),
+    c("-e", "--min-cells-expressed"),
     action = "store",
     default = 0,
-    type = 'numeric',
-    help = "Minimum number of expressed genes to filter cells on"
-  ),
-  make_option(
-    c("-m", "--percent-counts-MT"),
-    action = "store",
-    default = 100,
-    type = 'numeric',
-    help = "Maximum % of mitochondrial genes expressed per cell. Cells that exceed this value will be filtered out."
+    type = "numeric",
+    help = "Remove features that occur in less than the given number of cells"
   ),
   make_option(
     c("-o", "--output-loom"),
     action = "store",
     default = NA,
-    type = 'character',
-    help = "File name in which to store the SingleCellExperiment object in Loom format."
+    type = "character",
+    help = "File name in which to store the SingleCellExperiment object in Loom format"
   )
 )
 
-opt <- wsc_parse_args(option_list, mandatory = c('input_loom', 'output_loom'))
+opt <- wsc_parse_args(option_list, mandatory = c("input_loom", "output_loom"))
 
 # Check parameter values
 
-if ( ! file.exists(opt$input_loom)){
-  stop((paste('File', opt$input_loom, 'does not exist')))
+if (! file.exists(opt$input_loom)) {
+  stop((paste("File", opt$input_loom, "does not exist")))
 }
 
 # Filter out unexpressed features
 
-scle <- import(opt$input_loom, format='loom', type='SingleCellLoomExperiment')
-print(paste("Starting with", ncol(scle), "cells and", nrow(scle), "features."))
+sce <- import(opt$input_loom, format = "loom", type = "SingleCellLoomExperiment")
+print(paste("Starting with", ncol(sce), "cells and", nrow(sce), "features."))
 
-# Create a logical vector of features that are expressed (above detection_limit)
-feature_expressed <- nexprs(scle, detection_limit = opt$detection_limit, exprs_values = 1, byrow=TRUE) > 0
-scle <- scle[feature_expressed, ]
-
-print(paste("After filtering out unexpressed features: ", ncol(scle), "cells and", nrow(scle), "features."))
+# Filter out low quality cells
 
 # Filter low library sizes
-to_keep <- scle$total_counts > opt$library_size
-scle <- scle[, to_keep]
-
-print(paste("After filtering out low library counts: ", ncol(scle), "cells and", nrow(scle), "features."))
-
-
-# Filter low expressed genes
-to_keep <- scle$total_features_by_counts > opt$expressed_genes
-scle <- scle[, to_keep]
-
-print(paste("After filtering out low expressed: ", ncol(scle), "cells and", nrow(scle), "features."))
-
+passing_total <- sce$total > opt$library_size
+sce <- sce[, passing_total]
+print(paste("After filtering out low library counts: ", ncol(sce), "cells and", nrow(sce), "features."))
 
 # Filter out high MT counts
-to_keep <- scle$pct_counts_MT < opt$percent_counts_MT
-scle <- scle[, to_keep]
+passing_mt_counts <- sce$subsets_Mito_percent < opt$percent_counts_MT
+sce <- sce[, passing_mt_counts]
+print(paste("After filtering out high MT gene counts: ", ncol(sce), "cells and", nrow(sce), "features."))
 
-print(paste("After filtering out high MT gene counts: ", ncol(scle), "cells and", nrow(scle), "features."))
+expr_features <- sce$detected > opt$expressed_features
+sce <- sce[, expr_features]
+print(paste("After filtering out cells with low feature counts: ", ncol(sce), "cells and", nrow(sce), "features."))
+
+# Create a logical vector of features that are expressed (above detection_limit)
+feature_expressed <- nexprs(sce, detection_limit = opt$detection_limit, byrow = TRUE) > opt$min_cells_expressed
+sce <- sce[feature_expressed, ]
+print(paste("After filtering out rare features: ", ncol(sce), "cells and", nrow(sce), "features."))
 
 # Output to a Loom file
 if (file.exists(opt$output_loom)) {
   file.remove(opt$output_loom)
 }
-export(scle, opt$output_loom, format='loom')
+export(sce, opt$output_loom, format = "loom")
--- a/scater-normalize.R	Tue Sep 03 14:30:21 2019 -0400
+++ /dev/null	Thu Jan 01 00:00:00 1970 +0000
@@ -1,50 +0,0 @@
-#!/usr/bin/env Rscript
-#Normalises a SingleCellExperiment object
-
-# Load optparse we need to check inputs
-library(optparse)
-library(workflowscriptscommon)
-library(LoomExperiment)
-library(scater)
-
-# parse options
-option_list = list(
-  make_option(
-    c("-i", "--input-loom"),
-    action = "store",
-    default = NA,
-    type = 'character',
-    help = "A SingleCellExperiment object file in Loom format."
-  ),
-  make_option(
-    c("-o", "--output-loom"),
-    action = "store",
-    default = NA,
-    type = 'character',
-    help = "File name in which to store the SingleCellExperiment object in Loom format."
-  )
-)
-
-opt <- wsc_parse_args(option_list, mandatory = c('input_loom', 'output_loom'))
-
-# Check parameter values
-
-if ( ! file.exists(opt$input_loom)){
-  stop((paste('File', opt$input_loom, 'does not exist')))
-}
-
-# Input from Loom format
-
-scle <- import(opt$input_loom, format='loom', type='SingleCellLoomExperiment')
-print(paste("Normalising...."))
-
-#Normalise
-scle <- normalize(scle, exprs_values = 1)
-
-print(paste("Finished normalising"))
-
-# Output to a Loom file
-if (file.exists(opt$output_loom)) {
-  file.remove(opt$output_loom)
-}
-export(scle, opt$output_loom, format='loom')
--- a/scater-pca-filter.R	Tue Sep 03 14:30:21 2019 -0400
+++ b/scater-pca-filter.R	Thu Sep 09 12:24:39 2021 +0000
@@ -12,49 +12,48 @@
 library(workflowscriptscommon)
 library(LoomExperiment)
 library(scater)
-library(mvoutlier)
+library(robustbase)
 
 # parse options
-option_list = list(
+option_list <- list(
   make_option(
     c("-i", "--input-loom"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "A SingleCellExperiment object file in Loom format."
   ),
   make_option(
     c("-o", "--output-loom"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "File name in which to store the SingleCellExperiment object in Loom format."
   )
 )
 
-opt <- wsc_parse_args(option_list, mandatory = c('input_loom', 'output_loom'))
+opt <- wsc_parse_args(option_list, mandatory = c("input_loom", "output_loom"))
 
 # Check parameter values
 
-if ( ! file.exists(opt$input_loom)){
-  stop((paste('File', opt$input_loom, 'does not exist')))
+if (! file.exists(opt$input_loom)) {
+  stop((paste("File", opt$input_loom, "does not exist")))
 }
 
-# Input from Loom format
+# Filter out unexpressed features
 
-scle <- import(opt$input_loom, format='loom', type='SingleCellLoomExperiment')
-print(paste("Starting with", ncol(scle), "cells and", nrow(scle), "features."))
+sce <- import(opt$input_loom, format = "loom", type = "SingleCellLoomExperiment")
+
+print(paste("Starting with", ncol(sce), "cells"))
 
-# Run PCA on data and detect outliers
-scle <- runPCA(scle, use_coldata = TRUE, detect_outliers = TRUE)
+sce <- runColDataPCA(sce, outliers = TRUE, variables = list("sum", "detected", "subsets_Mito_percent"))
+sce$use <- !sce$outlier
+sce <- sce[, colData(sce)$use]
+print(paste("Ending with", ncol(sce), "cells"))
 
-# Filter out outliers
-scle <- scle[, !scle$outlier]
-
-print(paste("Ending with", ncol(scle), "cells and", nrow(scle), "features."))
 
 # Output to a Loom file
 if (file.exists(opt$output_loom)) {
   file.remove(opt$output_loom)
 }
-export(scle, opt$output_loom, format='loom')
+export(sce, opt$output_loom, format = "loom")
--- a/scater-plot-dist-scatter.R	Tue Sep 03 14:30:21 2019 -0400
+++ b/scater-plot-dist-scatter.R	Thu Sep 09 12:24:39 2021 +0000
@@ -13,45 +13,45 @@
 
 # parse options
 
-option_list = list(
+option_list <- list(
   make_option(
     c("-i", "--input-loom"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "A SingleCellExperiment object file in Loom format."
   ),
   make_option(
     c("-o", "--output-plot-file"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "Path of the PDF output file to save plot to."
   ),
   make_option(
     c("-l", "--log-scale"),
-    action="store_true",
-    default=FALSE,
-    type = 'logical',
+    action = "store_true",
+    default = FALSE,
+    type = "logical",
     help = "Plot on log scale (recommended for large datasets)."
   )
 )
 
-opt <- wsc_parse_args(option_list, mandatory = c('input_loom', 'output_plot_file', 'log_scale'))
+opt <- wsc_parse_args(option_list, mandatory = c("input_loom", "output_plot_file"))
 
 # Check parameter values
 
-if ( ! file.exists(opt$input_loom)){
-  stop((paste('File', opt$input_loom, 'does not exist')))
+if (! file.exists(opt$input_loom)) {
+  stop((paste("File", opt$input_loom, "does not exist")))
 }
 
-# Input from Loom format
+# Filter out unexpressed features
 
-scle <- import(opt$input_loom, format='loom', type='SingleCellLoomExperiment')
+sce <- import(opt$input_loom, format = "loom", type = "SingleCellLoomExperiment")
 
-#do the scatter plot of reads vs genes
-total_counts <- scle$total_counts
-total_features <- scle$total_features_by_counts
+# Do the scatter plot of reads vs genes
+total_counts <- sce$total
+total_features <- sce$detected
 count_feats <- cbind(total_counts, total_features)
 cf_dm <- as.data.frame(count_feats)
 
@@ -59,21 +59,20 @@
 read_bins <- max(total_counts / 1e6) / 20
 feat_bins <- max(total_features) / 20
 
-# Make the plots
-plot <- ggplot(cf_dm, aes(x=total_counts / 1e6, y=total_features)) + geom_point(shape=1) + geom_smooth() + xlab("Read count (millions)") +
-   ylab("Feature count") + ggtitle("Scatterplot of reads vs features")
-plot1 <- qplot(total_counts / 1e6, geom="histogram", binwidth = read_bins, ylab="Number of cells", xlab = "Read counts (millions)", fill=I("darkseagreen3")) + ggtitle("Read counts per cell")
-plot2 <- qplot(total_features, geom="histogram", binwidth = feat_bins, ylab="Number of cells", xlab = "Feature counts", fill=I("darkseagreen3")) + ggtitle("Feature counts per cell")
-plot3 <- plotColData(scle, y="pct_counts_MT", x="total_features_by_counts") + ggtitle("% MT genes") + geom_point(shape=1) + theme(text = element_text(size=15)) + theme(plot.title = element_text(size=15))
+plot1 <- qplot(total_counts / 1e6, geom = "histogram", binwidth = read_bins, ylab = "Number of cells", xlab = "Read counts (millions)", fill = I("darkseagreen3")) + ggtitle("Read counts per cell")
+plot2 <- qplot(total_features, geom = "histogram", binwidth = feat_bins, ylab = "Number of cells", xlab = "Feature counts", fill = I("darkseagreen3")) + ggtitle("Feature counts per cell")
+plot3 <- ggplot(cf_dm, aes(x = total_counts / 1e6, y = total_features)) + geom_point(shape = 1) + geom_smooth() + xlab("Read count (millions)") +
+  ylab("Feature count") + ggtitle("Scatterplot of reads vs features")
+plot4 <- plotColData(sce, y = "subsets_Mito_percent", x = "detected") + ggtitle("% MT genes") + geom_point(shape = 1) + theme(text = element_text(size = 15)) + theme(plot.title = element_text(size = 15)) + xlab("Total features") + ylab("% MT")
 
-if (! opt$log_scale){
-  final_plot <- ggarrange(plot1, plot2, plot, plot3, ncol=2, nrow=2)
-  ggsave(opt$output_plot_file, final_plot, device="pdf")
+if (! opt$log_scale) {
+  final_plot <- ggarrange(plot1, plot2, plot3, plot4, ncol = 2, nrow = 2)
+  ggsave(opt$output_plot_file, final_plot, device = "pdf")
 } else {
-  plot_log_both <- plot + scale_x_continuous(trans = 'log10') + scale_y_continuous(trans = 'log10')
-  plot1_log <- plot1 + scale_y_continuous(trans = 'log10')
-  plot2_log <- plot2 + scale_y_continuous(trans = 'log10')
-  plot3_log <- plot3 + scale_y_log10(labels=number)
-  final_plot_log <- ggarrange(plot1_log, plot2_log, plot_log_both, plot3_log, ncol=2, nrow=2)
-  ggsave(opt$output_plot_file, final_plot_log, device="pdf")
+  plot1_log <- plot1 + scale_x_continuous(trans = "log10") + scale_y_continuous(trans = "log10")
+  plot2_log <- plot2 + scale_y_continuous(trans = "log10")
+  plot3_log <- plot3 + scale_y_continuous(trans = "log10")
+  plot4_log <- plot4 + scale_y_log10(labels = number)
+  final_plot_log <- ggarrange(plot1_log, plot2_log, plot3_log, plot4_log, ncol = 2, nrow = 2)
+  ggsave(opt$output_plot_file, final_plot_log, device = "pdf")
 }
--- a/scater-plot-exprs-freq.R	Tue Sep 03 14:30:21 2019 -0400
+++ /dev/null	Thu Jan 01 00:00:00 1970 +0000
@@ -1,45 +0,0 @@
-#!/usr/bin/env Rscript
-
-#Plots mean expression vs % of expressing cells and provides information as to the number of genes expressed in 50% and 25% of cells.
-# Load optparse we need to check inputs
-
-library(optparse)
-library(workflowscriptscommon)
-library(LoomExperiment)
-library(scater)
-
-# parse options
-
-option_list = list(
-  make_option(
-    c("-i", "--input-loom"),
-    action = "store",
-    default = NA,
-    type = 'character',
-    help = "A SingleCellExperiment object file in Loom format."
-  ),
-  make_option(
-    c("-o", "--output-plot-file"),
-    action = "store",
-    default = NA,
-    type = 'character',
-    help = "Path of the PDF output file to save plot to."
-  )
-)
-
-opt <- wsc_parse_args(option_list, mandatory = c('input_loom', 'output_plot_file'))
-
-# Check parameter values
-
-if ( ! file.exists(opt$input_loom)){
-  stop((paste('File', opt$input_loom, 'does not exist')))
-}
-
-
-# Input from Loom format
-
-scle <- import(opt$input_loom, format='loom', type='SingleCellLoomExperiment')
-
-#produce and save the scatter plot of reads vs genes
-plot <- plotExprsFreqVsMean(scle, controls = "is_feature_control_MT")
-ggsave(opt$output_plot_file, plot, device="pdf")
--- a/scater-plot-pca.R	Tue Sep 03 14:30:21 2019 -0400
+++ b/scater-plot-pca.R	Thu Sep 09 12:24:39 2021 +0000
@@ -11,58 +11,58 @@
 
 # parse options
 
-option_list = list(
+option_list <- list(
   make_option(
     c("-i", "--input-loom"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "A SingleCellExperiment object file in Loom format."
   ),
   make_option(
     c("-c", "--colour-by"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Feature (from annotation file) to colour PCA plot points by. The values represented in this options should be categorical"
   ),
   make_option(
     c("-s", "--size-by"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Feature (from annotation file) to size PCA plot points by. The values represented in this options should be numerical and not categorical"
   ),
   make_option(
     c("-p", "--shape-by"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Feature (from annotation file) to shape PCA plot points by. The values represented in this options should be categorical"
   ),
   make_option(
     c("-o", "--output-plot-file"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "Path of the PDF output file to save plot to."
   )
 )
 
-opt <- wsc_parse_args(option_list, mandatory = c('input_loom', 'output_plot_file'))
+opt <- wsc_parse_args(option_list, mandatory = c("input_loom", "output_plot_file"))
+
 # Check parameter values
 
-if ( ! file.exists(opt$input_loom)){
-  stop((paste('File', opt$input_loom, 'does not exist')))
+if (! file.exists(opt$input_loom)) {
+  stop((paste("File", opt$input_loom, "does not exist")))
 }
 
-
-# Input from Loom format
+# Filter out unexpressed features
 
-scle <- import(opt$input_loom, format='loom', type='SingleCellLoomExperiment')
-scle <- normalize(scle, exprs_values = 1)
-scle <- runPCA(scle)
-plot <- plotReducedDim(scle, "PCA", colour_by = opt$colour_by, size_by = opt$size_by, shape_by = opt$shape_by)
-#do the scatter plot of reads vs genes
+sce <- import(opt$input_loom, format = "loom", type = "SingleCellLoomExperiment")
+sce <- logNormCounts(sce)
+sce <- runPCA(sce)
 
-ggsave(opt$output_plot_file, plot, device="pdf")
+plot <- plotReducedDim(sce, dimred = "PCA", colour_by = opt$colour_by, size_by = opt$size_by, shape_by = opt$shape_by)
+
+ggsave(opt$output_plot_file, plot, device = "pdf")
--- a/scater-plot-tsne.R	Tue Sep 03 14:30:21 2019 -0400
+++ b/scater-plot-tsne.R	Thu Sep 09 12:24:39 2021 +0000
@@ -12,58 +12,57 @@
 
 # parse options
 
-option_list = list(
+option_list <- list(
   make_option(
     c("-i", "--input-loom"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "A SingleCellExperiment object file in Loom format."
   ),
   make_option(
     c("-c", "--colour-by"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Feature (from annotation file) to colour t-SNE plot points by. The values represented in this options should be categorical"
   ),
   make_option(
     c("-s", "--size-by"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Feature (from annotation file) to size t-SNE plot points by. The values represented in this options should be numerical and not categorical"
   ),
   make_option(
     c("-p", "--shape-by"),
     action = "store",
     default = NULL,
-    type = 'character',
+    type = "character",
     help = "Feature (from annotation file) to shape t-SNE plot points by. The values represented in this options should be categorical"
   ),
   make_option(
     c("-o", "--output-plot-file"),
     action = "store",
     default = NA,
-    type = 'character',
+    type = "character",
     help = "Path of the PDF output file to save plot to."
   )
 )
 
-opt <- wsc_parse_args(option_list, mandatory = c('input_loom', 'output_plot_file'))
+opt <- wsc_parse_args(option_list, mandatory = c("input_loom", "output_plot_file"))
+
 # Check parameter values
 
-if ( ! file.exists(opt$input_loom)){
-  stop((paste('File', opt$input_loom, 'does not exist')))
+if (! file.exists(opt$input_loom)) {
+  stop((paste("File", opt$input_loom, "does not exist")))
 }
 
-
-# Input from Loom format
+# Filter out unexpressed features
 
-scle <- import(opt$input_loom, format='loom', type='SingleCellLoomExperiment')
-scle <- normalize(scle, exprs_values = 1)
-scle <- runTSNE(scle, perplexity=10)
-plot <- plotTSNE(scle, colour_by = opt$colour_by, size_by = opt$size_by, shape_by = opt$shape_by)
+sce <- import(opt$input_loom, format = "loom", type = "SingleCellLoomExperiment")
+sce <- logNormCounts(sce)
+sce <- runTSNE(sce, perplexity = 10)
+plot <- plotTSNE(sce, colour_by = opt$colour_by, size_by = opt$size_by, shape_by = opt$shape_by)
 
-
-ggsave(opt$output_plot_file, plot, device="pdf")
+ggsave(opt$output_plot_file, plot, device = "pdf")
--- a/scater-plot-tsne.xml	Tue Sep 03 14:30:21 2019 -0400
+++ b/scater-plot-tsne.xml	Thu Sep 09 12:24:39 2021 +0000
@@ -1,5 +1,6 @@
-<tool id="scater_plot_tsne" name="Scater: t-SNE plot" version="@TOOL_VERSION@">
-    <description>Produces a t-SNE plot of two components</description>
+<tool id="scater_plot_tsne" name="Scater: t-SNE plot" version="@TOOL_VERSION@" profile="@PROFILE@">
+    <description>of two components</description>
+    <expand macro="bio_tools"/>
     <macros>
         <import>macros.xml</import>
     </macros>
Binary file test-data/scater_exprs_freq.pdf has changed
Binary file test-data/scater_filtered_normalised.loom has changed
Binary file test-data/scater_manual_filtered.loom has changed
Binary file test-data/scater_pca_filtered.loom has changed
Binary file test-data/scater_pca_plot.pdf has changed
Binary file test-data/scater_qcready.loom has changed
Binary file test-data/scater_reads_genes_dist.pdf has changed
Binary file test-data/scater_reads_genes_dist_log.pdf has changed
Binary file test-data/scater_tsne_plot.pdf has changed