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 "cells": [
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "# IDR0021 - ROIs"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "A simple example which reads the 'batch_roi_export' table, performs some basic statistics on it and creates a plot (which should be similar to [Figure 1](https://www.nature.com/articles/ncb2591/figures/1) in the paper ['Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material'](https://doi.org/10.1038/ncb2591)). The plot and results are then uploaded to the server and attached to the project."
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Load the romero.gateway library and connect to the server"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {},
   "outputs": [],
   "source": [
    "options(warn=-1) # Disable warnings to suppress unnecessary output\n",
    "\n",
    "library(romero.gateway)\n",
    "\n",
    "user_name = readline('Username: ')\n",
    "user_password <- getPass::getPass('OMERO password: ')\n",
    "server <- OMEROServer(host = 'wss://workshop.openmicroscopy.org/omero-ws', port = 443L, username = user_name, password = user_password)\n",
    "server <- connect(server)\n",
    "paste('Successfully logged in as', server@user$getUserName())"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Load the data from the 'batch_roi_export' table as R dataframe"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {},
   "outputs": [],
   "source": [
    "# Project with the 'Summary_from_Fiji' table attached\n",
    "projectId <- 872\n",
    "tableName <- 'Summary_from_Fiji'\n",
    "\n",
    "project <- loadObject(server, \"ProjectData\", projectId)\n",
    "\n",
    "annos <- getAnnotations(server, 'ProjectData', projectId, nameFilter = tableName)\n",
    "annotationFileID = as.integer(annos$FileID)\n",
    "\n",
    "# Load the table (column 14 and 15, Dataset and bouding_box) directly as R dataframe\n",
    "df <- loadDataframe(project, annotationFileID, columns=c(14, 15))\n",
    "df"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Prepare the data"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {},
   "outputs": [],
   "source": [
    "# CEP120 is represented by two datasets'CEP120/20111106' and 'CEP120/20111209',\n",
    "# just combine them to 'CEP120'\n",
    "df$Dataset <- as.character(df$Dataset)\n",
    "df[ df == 'CEP120/20111106' ] <- 'CEP120'\n",
    "df[ df == 'CEP120/20111209' ] <- 'CEP120'\n",
    "df$Dataset <- as.factor(df$Dataset)\n",
    "\n",
    "\n",
    "# Use the bounding box of the biggest shapes for the specified channel\n",
    "df$diameter <- sqrt(df$Bounding_Box)\n",
    "df"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Plot the data"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {},
   "outputs": [],
   "source": [
    "# Order the data from lowest to highest mean diameter (to match the order of figure 1)\n",
    "ag <-aggregate(df$diameter ~ df$Dataset, df, mean)\n",
    "orderedDatasets <- factor(df$Dataset, levels=ag[order(ag$`df$diameter`), 'df$Dataset'])\n",
    "\n",
    "plot(df$diameter ~ orderedDatasets, ylab='Bounding Box', xlab=\"Protein\", cex.axis=0.5)"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Is there a significant difference between the proteins?"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {
    "scrolled": true
   },
   "outputs": [],
   "source": [
    "# One-way analysis of variance:\n",
    "fit <- aov(df$diameter ~ df$Dataset)\n",
    "summary(fit)\n",
    "\n",
    "# Two-sample Wilcoxon test ('Mann-Whitney') of all pairwise combinations:\n",
    "combins <- combn(levels(df$Dataset), 2)\n",
    "params_list <- split(as.vector(combins), rep(1:ncol(combins), each = nrow(combins)))\n",
    "testResults <- data.frame()\n",
    "for (param in params_list) {\n",
    "  testdf <- subset(df, df$Dataset %in% param)\n",
    "  pval <- wilcox.test(formula = diameter ~ Dataset, data = testdf)$p.value\n",
    "  testResults<-rbind(testResults, data.frame(Protein_1=param[1], Protein_2=param[2], p_value=pval))\n",
    "}\n",
    "testResults"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "## Attach the results to OMERO project and close connection"
   ]
  },
  {
   "cell_type": "code",
   "execution_count": null,
   "metadata": {},
   "outputs": [],
   "source": [
    "# The plot:\n",
    "tmpfile <- \"/tmp/Bounding_Box_By_Protein.png\"\n",
    "png(tmpfile, width = 4, height= 2.5, units = \"in\", res = 300, pointsize = 6)\n",
    "plot(df$diameter ~ orderedDatasets, ylab='Bounding Box', xlab=\"Protein\", cex.axis=0.5)\n",
    "dev.off()\n",
    "invisible(attachFile(project, tmpfile)) # Wrapped in invisible(...) only to suppress unnecessary output\n",
    "\n",
    "\n",
    "# The results from the Mann-Whitney test as OMERO.table (HDF)\n",
    "invisible(attachDataframe(project, testResults, \"Mann-Whitney-pValues\"))\n",
    "\n",
    "# and as CSV file:\n",
    "tmpfile <- \"/tmp/Mann-Whitney-pValues.csv\"\n",
    "write.csv(testResults, file = tmpfile)\n",
    "invisible(attachFile(project, tmpfile)) \n",
    "\n",
    "# Finally disconnect again\n",
    "invisible(disconnect(server))"
   ]
  },
  {
   "cell_type": "markdown",
   "metadata": {},
   "source": [
    "### License (BSD 2-Clause)\n",
    "\n",
    "Copyright (c) 2021, University of Dundee All rights reserved.\n",
    "\n",
    "Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:\n",
    "\n",
    "Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.\n",
    "Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.\n",
    "THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS \"AS IS\" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT OWNER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE."
   ]
  }
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