--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/seurat.R	Sun Aug 26 16:24:02 2018 -0400
@@ -0,0 +1,116 @@
+options( show.error.messages=F, error = function () { cat( geterrmessage(), file=stderr() ); q( "no", 1, F ) } )
+
+# we need that to not crash galaxy with an UTF8 error on German LC settings.
+loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8")
+
+suppressPackageStartupMessages({
+    library(Seurat)
+    library(SingleCellExperiment)
+    library(dplyr)
+    library(optparse)
+})
+
+option_list <- list(
+    make_option(c("-counts","--counts"), type="character", help="Counts file"),
+    make_option(c("-numPCs","--numPCs"), type="integer", help="Number of PCs to use in plots"),
+    make_option(c("-min.cells","--min.cells"), type="integer", help="Minimum cells to include"),
+    make_option(c("-min.genes","--min.genes"), type="integer", help="Minimum genes to include"),
+    make_option(c("-low.thresholds","--low.thresholds"), type="double", help="Low threshold for filtering cells"),
+    make_option(c("-high.thresholds","--high.thresholds"), type="double", help="High threshold for filtering cells"),
+    make_option(c("-x.low.cutoff","--x.low.cutoff"), type="double", help="X-axis low cutoff for variable genes"),
+    make_option(c("-x.high.cutoff","--x.high.cutoff"), type="double", help="X-axis high cutoff for variable genes"),
+    make_option(c("-y.cutoff","--y.cutoff"), type="double", help="Y-axis cutoff for variable genes"),
+    make_option(c("-cells.use","--cells.use"), type="integer", help="Cells to use for PCHeatmap"),
+    make_option(c("-resolution","--resolution"), type="double", help="Resolution in FindClusters"),
+    make_option(c("-min.pct","--min.pct"), type="double", help="Minimum percent cells in FindClusters"),
+    make_option(c("-logfc.threshold","--logfc.threshold"), type="double", help="LogFC threshold in FindClusters"),
+    make_option(c("-rds","--rds"), type="logical", help="Output Seurat RDS object")
+  )
+
+parser <- OptionParser(usage = "%prog [options] file", option_list=option_list)
+args = parse_args(parser)
+
+counts <- read.delim(args$counts, row.names=1)
+seuset <- CreateSeuratObject(raw.data = counts, min.cells = args$min.cells, min.genes = args$min.cells)
+
+# Open PDF for plots
+pdf("out.pdf")
+
+VlnPlot(object = seuset, features.plot = c("nGene", "nUMI"), nCol = 2)
+GenePlot(object = seuset, gene1 = "nUMI", gene2 = "nGene")
+
+print("Filtering cells")
+if (!is.null(args$low.thresholds)){
+    lowthresh <- args$low.thresholds
+} else {
+    lowthresh <- "-Inf"
+}
+if (!is.null(args$high.thresholds)){
+    highthresh <- args$high.thresholds
+} else {
+    highthresh <- "Inf"
+}
+seuset <- FilterCells(object = seuset, subset.names = c("nUMI"), 
+    low.thresholds=c(lowthresh), high.thresholds = c(highthresh))
+
+print("Normalizing the data")
+seuset <- NormalizeData(object = seuset, normalization.method = "LogNormalize", 
+    scale.factor = 10000)
+
+print("Finding variable genes")
+seuset <- FindVariableGenes(object = seuset, mean.function = ExpMean, 
+    dispersion.function = LogVMR, 
+    x.low.cutoff = args$x.low.cutoff, 
+    x.high.cutoff = args$x.high.cutoff,,
+    y.cutoff = args$y.cutoff
+)
+
+print("Scaling the data and removing unwanted sources of variation")
+seuset <- ScaleData(object = seuset, vars.to.regress = c("nUMI"))
+
+print("Performing PCA analysis")
+seuset <- RunPCA(object = seuset, pc.genes = seuset@var.genes)
+VizPCA(object = seuset, pcs.use = 1:2)
+PCAPlot(object = seuset, dim.1 = 1, dim.2 = 2)
+PCHeatmap(
+    object = seuset, 
+    pc.use = 1:args$numPCs, 
+    cells.use = args$cell.use, 
+    do.balanced = TRUE, 
+    label.columns = FALSE,
+    use.full = FALSE
+)
+
+print("Determining statistically significant principal components")
+seuset <- JackStraw(object = seuset, num.replicate = 100, display.progress= FALSE)
+JackStrawPlot(object = seuset, PCs = 1:args$numPCs)
+PCElbowPlot(object = seuset)
+
+print("Clustering the cells")
+seuset <- FindClusters(
+    object = seuset, 
+    reduction.type = "pca", 
+    dims.use = 1:args$numPCs, 
+    resolution = args$resolution,
+    print.output = 0, 
+    save.SNN = TRUE
+)
+
+print("Running non-linear dimensional reduction (tSNE)")
+seuset <- RunTSNE(object = seuset, dims.use = 1:args$numPCs, do.fast = TRUE)
+TSNEPlot(object = seuset)
+
+print("Finding differentially expressed genes (cluster biomarkers)")
+markers <- FindAllMarkers(object = seuset, only.pos = TRUE, min.pct = args$min.pct,
+    logfc.threshold = args$logfc.threshold)
+top10 <- markers %>% group_by(cluster) %>% top_n(10, avg_logFC)
+DoHeatmap(object = seuset, genes.use = top10$gene, slim.col.label = TRUE, remove.key = TRUE)
+
+# Close PDF for plots
+dev.off()
+
+if (!is.null(args$rds) ) {
+  saveRDS(seuset, "Seurat.rds")
+}
+
+sessionInfo()
\ No newline at end of file

--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/seurat.xml	Sun Aug 26 16:24:02 2018 -0400
@@ -0,0 +1,146 @@
+<tool id="seurat" name="Seurat" version="2.3.4">
+    <description>- toolkit for exploration of single-cell RNA-seq data</description>
+    <requirements>
+        <requirement type="package" version="3.4.1">r-base</requirement>
+        <requirement type="package" version="2.3.4">r-seurat</requirement>
+        <requirement type="package" version="1.0.0">bioconductor-singlecellexperiment</requirement> 
+        <requirement type="package" version="1.6.0">r-optparse</requirement>
+    </requirements>
+    <version_command><![CDATA[
+echo $(R --version | grep version | grep -v GNU)", seurat version" $(R --vanilla --slave -e "library(seurat); cat(sessionInfo()\$otherPkgs\$seurat\$Version)" 2> /dev/null | grep -v -i "WARNING: ")", SingleCellExperiment version" $(R --vanilla --slave -e "library(SingleCellExperiment); cat(sessionInfo()\$otherPkgs\$SingleCellExperiment\$Version)" 2> /dev/null | grep -v -i "WARNING: ")", optparse version" $(R --vanilla --slave -e "library(optparse); cat(sessionInfo()\$otherPkgs\$optparse\$Version)" 2> /dev/null | grep -v -i "WARNING: ")
+    ]]></version_command>
+    <command detect_errors="exit_code"><![CDATA[
+
+#if $rscript:
+    cp '$__tool_directory__/seurat.R' '$out_rscript' &&
+#end if
+
+Rscript '$__tool_directory__/seurat.R'
+
+--counts '$counts'
+--numPCs $adv.num_PCs
+--min.cells $adv.min_cells
+--min.genes $adv.min_genes
+
+#if $adv.low_thresholds:
+    --low.thresholds $adv.low_thresholds
+#end if
+#if $adv.high_thresholds:
+    --high.thresholds $adv.high_thresholds
+#end if
+#if $adv.x_low_cutoff:
+    --x.low.cutoff $adv.x_low_cutoff
+#end if
+#if $adv.x_high_cutoff:
+    --x.high.cutoff $adv.x_high_cutoff
+#end if
+#if $adv.y_cutoff:
+    --y.cutoff $adv.y_cutoff
+#end if
+#if $adv.cells_use:
+    --cells.use $adv.cells_use
+#end if
+#if $adv.resolution:
+    --resolution $adv.resolution
+#end if
+#if $adv.min_pct:
+    --min.pct $adv.min_pct
+#end if
+#if $adv.logfc_threshold:
+    --logfc.threshold $adv.logfc_threshold
+#end if
+
+#if $rds:
+    --rds '$rds'
+#end if
+
+]]></command>
+
+    <inputs>
+        <param name="counts" type="data" format="tabular" label="Counts file" help="The should be a TAB-separated count matrix with gene identifers in the first column and a header row"/>
+        <param name="rscript" type="boolean" truevalue="TRUE" falsevalue="FALSE" checked="False" label="Output Rscript?" help="If this option is set to Yes, the Rscript used by this tool will be provided as a text file in the output. Default: No" />
+        <param name="rds" type="boolean" truevalue="True" falsevalue="False" checked="False" label="Output Seurat RDS object?"
+            help="Output the Seurat RDS object, can be loaded into R. Default: No">
+        </param>
+        <section name="adv" title="Advanced Options">
+            <param argument="--num_PCs" type="integer" min="0" value="10" label="Number of PCs to use in plots" help="Uses this number of PCs in PCHEatmap, JackStrawPlot, FindClusters, RunTSNE. Default: 10" />
+            <param argument="--min_cells" type="integer" min="0" value="0" label="Minimum cells" help="Include genes with detected expression in at least this many cells." />
+            <param argument="--min_genes" type="integer" min="0"  value="0" label="Minimum genes" help="Include cells where at least this many genes are detected." />
+            <param argument="--low_thresholds" type="float" optional="True" label="Low threshold for filtering cells" />
+            <param argument="--high_thresholds" type="float" optional="True" label="High threshold for filtering cells" />
+            <param argument="--x_low_cutoff" type="float" optional="True" label="X-axis low cutoff for variable genes" help="Bottom cutoff on x-axis for identifying variable genes" />
+            <param argument="--x_high_cutoff" type="float" optional="True" label="X-axis high cutoff for variable genes" help="Top cutoff on x-axis for identifying variable genes" />
+            <param argument="--y_cutoff" type="float" optional="True" label="Y-axis cutoff for variable genes" help="Bottom cutoff on y-axis for identifying variable genes" />
+            <param argument="--cells_use" type="integer" min="0" optional="True" label="Cells to use for PCHeatmap" help="Plots this number of top ‘extreme’ cells on both ends of the spectrum, which dramatically speeds plotting for large datasets" />
+            <param argument="--resolution" type="float" optional="True" label="Resolution parameter" help="Value of the resolution parameter used in FindClusters, use a value above (below) 1.0 if you want to obtain a larger (smaller) number of communities." />
+            <param argument="--min_pct" type="float" optional="True" label="Minimum percent cells" help="With FindMarkers only test genes that are detected in a minimum fraction of min.pct cells in either of the two populations. Meant to speed up the function by not testing genes that are very infrequently expressed. Default is 0.1" />
+            <param argument="--logfc_threshold" type="float" min="0" optional="True" label="LogFC threshold"
+                help="With FindMarkers, limit testing to genes which show, on average, at least X-fold difference (log-scale)between the two groups of cells. Default is 0.25 Increasing logfc.threshold speeds up the function, but can miss weaker signals." />
+        </section>
+    </inputs>
+
+    <outputs>
+        <data name="out_pdf" format="pdf" from_work_dir="out.pdf" label="${tool.name} on ${on_string}: Plots" />
+        <data name="out_rscript" format="txt" from_work_dir="out_rscript.txt" label="${tool.name} on ${on_string}: Rscript">
+            <filter>rscript</filter>
+        </data>
+        <data name="out_rds" format="rds" from_work_dir="Seurat.rds" label="${tool.name} on ${on_string}: RData file">
+            <filter>rds</filter>
+        </data>
+    </outputs>
+
+    <tests>
+        <!-- Ensure count matrix input works -->
+        <test>
+            <param name="counts" ftype="tabular" value="deng_small.tab.gz"/>
+            <param name="min_cells" value="3"/>
+            <param name="min_genes" value="200"/>
+            <param name="low_thresholds" value="1" />
+            <param name="high_thresholds" value="20000000" />
+            <param name="x_low_cutoff" value="0.0125" />
+            <param name="x_high_cutoff" value="3" />
+            <param name="y_cutoff" value="0.5" />
+            <param name="numPCs" value="10" />
+            <param name="cells_use" value="500"/>
+            <param name="resolution" value="0.6" />
+            <param name="min_pct" value="0.25" />
+            <param name="logfc_threshold" value="0.25" />
+            <output name="out_pdf" ftype="pdf" value="out.pdf" compare="sim_size"/>
+        </test>
+    </tests>
+    <help><![CDATA[
+.. class:: infomark
+
+**What it does**
+
+Seurat_ is a toolkit for quality control, analysis, and exploration of single cell RNA sequencing data.
+It is developed and maintained by the `Satija Lab`_ at NYGC. Seurat aims to enable users to identify and
+interpret sources of heterogeneity from single cell transcriptomic measurements, and to integrate diverse
+types of single cell data. See the `Seurat Guided Clustering tutorial`_ for more information.
+
+-----
+
+**Inputs**
+
+    * Gene count matrix in TAB-separated format
+
+-----
+
+**Outputs**
+
+    * PDF of plots
+
+Optionally you can choose to output
+
+    * Seurat RDS object (can use within R)
+    * Rscript
+
+.. _Seurat: https://www.nature.com/articles/nbt.4096
+.. _Satija Lab: https://satijalab.org/seurat/
+.. _Seurat Guided Clustering tutorial: https://satijalab.org/seurat/pbmc3k_tutorial.html
+
+]]></help>
+    <citations>
+        <citation type="doi">10.1038/nbt.4096</citation>
+    </citations>
+</tool>

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