{ "cells": [ { "cell_type": "markdown", "id": "ef8532ee-d3b3-4227-b254-72302802ca5d", "metadata": {}, "source": [ "# deltact" ] }, { "cell_type": "markdown", "id": "2dfbb800-4a48-42fe-8333-29e7d8b224ef", "metadata": {}, "source": [ "ΔΔCt is the most common metric for quantifying gene expression changes by qPCR. Say you have a group of genes that you hypothesize varies between an experimental sample and a control. In both samples, you perform qPCR for both the genes of interest and some genes that you expect to be constant between the samples. These constant genes, often called housekeeping genes, are essential to the viability of the cell. 18S rRNA, RNA polymerase II, and GAPDH are common examples.\n", "\n", "To normalize for differences in cDNA input, subtract the average housekeeping gene Ct from the gene of interest Ct to get the ΔCt:\n", "\n", "$$\n", "Ct_{gene \\: of \\: interest} - Ct_{housekeeping} = \\Delta Ct\n", "$$\n", "\n", "Performing this calculation for both the experimental and control samples leaves you with two Ct values for each gene of interest:\n", "\n", "$$\n", "\\Delta Ct_{experimental} \\\\\n", "\\Delta Ct_{control}\n", "$$\n", "\n", "Subtracting the control ΔCt from the experimental yields the ΔΔCt:\n", "\n", "$$\n", "\\Delta Ct_{experimental} - \\Delta Ct_{control} = \\Delta \\Delta Ct\n", "$$\n", "\n", "The ΔΔCt is then the normalized cycle difference of the gene of interest between the experimental and control sample. Assuming that the efficiencies of all the primers are approximately 1, the ΔΔCt can be used to calculate fold change in gene expression:\n", "\n", "$$\n", "Fold \\: change = 2^{- \\Delta \\Delta Ct}\n", "$$\n", "\n", "So say that the ΔΔCt value is 1. This indicates that the PCR reaction in the experimental sample took 1 additional cycle to achieve the second derivative maximum than the control sample. Assuming perfect doubling with each cycle, the experimental sample should have half as much of the target transcript as the control:\n", "\n", "$$\n", "2^{-1} = 0.5\n", "$$\n", "\n", "It is worth emphasizing that this method is **semiquantitative**, as it assumes primer efficiencies of 1. [More advanced mathematical models](https://pubmed.ncbi.nlm.nih.gov/11328886/) than equipt currently uses can incorporate empirically determined efficiency values to gain true quantitative gene expression ratios. The \"Further Reading\" section of this document provides additional information on the complexities of relative gene expression using qPCR." ] }, { "cell_type": "markdown", "id": "8490b97a-7f71-4484-a5c7-26a7b630b800", "metadata": {}, "source": [ "## Importing data with namer" ] }, { "cell_type": "markdown", "id": "58df49ee-c2c8-4a3d-bc76-38873a3a4d88", "metadata": {}, "source": [ "As with all equipt tools, using `deltact()` begins with labeling the data using `namer()`. For this example, we will examine a dataset for siRNA validation of four genes:" ] }, { "cell_type": "code", "execution_count": 2, "id": "175d1dcd-b86a-4c40-bcdd-fed2bc477f62", "metadata": { "tags": [] }, "outputs": [], "source": [ "import equipt" ] }, { "cell_type": "code", "execution_count": 3, "id": "62406ebe-e213-4734-8081-76e522f3b0b0", "metadata": { "tags": [] }, "outputs": [ { "data": { "text/html": [ "
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PosCpPrimerNameNamePrim
0A111.2318SScramble-1Scramble-118S
1A211.2018SScramble-1Scramble-118S
2A311.2218SScramble-1Scramble-118S
3A411.2918SScramble-2Scramble-218S
4A511.3118SScramble-2Scramble-218S
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" ], "text/plain": [ " Pos Cp Primer Name NamePrim\n", "0 A1 11.23 18S Scramble-1 Scramble-118S\n", "1 A2 11.20 18S Scramble-1 Scramble-118S\n", "2 A3 11.22 18S Scramble-1 Scramble-118S\n", "3 A4 11.29 18S Scramble-2 Scramble-218S\n", "4 A5 11.31 18S Scramble-2 Scramble-218S" ] }, "execution_count": 3, "metadata": {}, "output_type": "execute_result" } ], "source": [ "primers = ('18S','Polr2a','Hdac3','Fus','Ewsr1','Taf15')\n", "\n", "samples = ['Scramble-1','Scramble-2',\n", " 'Hdac3-1','Hdac3-2',\n", " 'Fus-1','Fus-2',\n", " 'Taf15-1',\n", " 'Ewsr1-1','Ewsr1-2',\n", " 'Taf15-2']\n", "reps=3\n", "\n", "df = equipt.namer('data/23.01.31_FET-siRNAs_Ct.csv',\n", " primers,\n", " samples,\n", " reps,\n", " config='line',\n", " )\n", "\n", "df.head()" ] }, { "cell_type": "markdown", "id": "31f2f2b5-b9cb-46f0-a251-2aaba23a6b38", "metadata": {}, "source": [ "In this case, each sample had two biological replicates. Equipt does not currently perform statistical analyses, but having an indicator of the biological replicate in the samples list is required to prevent equipt from merging them into a single sample." ] }, { "cell_type": "markdown", "id": "134e652f-243c-4439-9ebc-1f9084445375", "metadata": {}, "source": [ "## Using deltact" ] }, { "cell_type": "markdown", "id": "efbbaad9-32a8-4529-ae73-a060a7a34d30", "metadata": {}, "source": [ "`deltact()` takes seven parameters. Their documentation is reproduced here:\n", "\n", " Params\n", " ______\n", " \n", " ct_data : a dataframe\n", " Output of namer().\n", " \n", " housekeeping : list\n", " A list of housekeeping primers.\n", " \n", " reps : int\n", " The number of replicate wells per sample-primer pair in the experiment.\n", " \n", " dilution : int or None\n", " If all samples have the same dilution factor, set to None. If samples\n", " have different dilution factors, set to the dilution factor of samples\n", " to be included in the dCt analysis. Default None\n", " \n", " thresh : float or None\n", " Highest acceptable standard deviation of replicate wells. If None, no \n", " thresholding is performed. If float, divergent wells are removed until\n", " replicate wells have acceptable deviation, and the number of removed \n", " wells for each sample primer pair is sent to droppedWells.txt. \n", " \n", " exp_ctrl : dictionary or None\n", " A dictionary containing pairs of experimental and control samples for\n", " ddCt analysis, e.g. {'+Drug1':'DMSO','+Drug2':'DMSO'}. If None, only\n", " dCt will be returned.\n", " \n", " foldchange : Bool\n", " Whether to convert ddCt values to fold change. Requires a valid\n", " dictionary for exp_ctrl.\n", " \n", "The function has a single output, a dataframe containing the requested values:\n", "\n", " Returns\n", " _______\n", " \n", " output : a dataframe\n", " A Pandas DataFrame containing the specified calculations.\n", " \n", " " ] }, { "cell_type": "markdown", "id": "8b9d7ba8-8c12-420f-a023-cf17797e8fe9", "metadata": {}, "source": [ "## ΔCt analysis" ] }, { "cell_type": "markdown", "id": "4e6bb1d2-abe9-4b83-9fca-339a7fe33a5e", "metadata": {}, "source": [ "In some cases, only the ΔCt value is necessary for analysis. For example, when using qPCR for restriction fragment length polymorphism (RFLP) analysis, a ΔCt value comparing plus and minus restriction enzyme in the same sample is often sufficient. As such, equipt has the option to stop after calculating ΔCt:" ] }, { "cell_type": "code", "execution_count": 5, "id": "f528461e-7164-491b-8b51-018fb50dc86e", "metadata": { "tags": [] }, "outputs": [ { "data": { "text/html": [ "
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NamePrimerdCtStdErr
0Ewsr1-1Ewsr14.4550000.112151
1Ewsr1-1Fus2.9816670.057155
2Ewsr1-1Hdac36.7183330.061644
3Ewsr1-1Taf155.3950000.071957
4Ewsr1-2Ewsr14.8083330.029627
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" ], "text/plain": [ " Name Primer dCt StdErr\n", "0 Ewsr1-1 Ewsr1 4.455000 0.112151\n", "1 Ewsr1-1 Fus 2.981667 0.057155\n", "2 Ewsr1-1 Hdac3 6.718333 0.061644\n", "3 Ewsr1-1 Taf15 5.395000 0.071957\n", "4 Ewsr1-2 Ewsr1 4.808333 0.029627" ] }, "execution_count": 5, "metadata": {}, "output_type": "execute_result" } ], "source": [ "# Identify reference genes \n", "housekeeping = ['18S','Polr2a']\n", "\n", "dct = equipt.deltact(df,\n", " housekeeping,\n", " reps,\n", " dilution=None,\n", " thresh=0.1,\n", " exp_ctrl=None,\n", " foldchange=False \n", " )\n", "\n", "dct.head()" ] }, { "cell_type": "markdown", "id": "48454d1d-8b3c-4a41-87a8-db4377bdf1c2", "metadata": {}, "source": [ "In this dataframe, we can see that for each sample a ΔCt value has been calculated between the indicated primer and the arithmetic mean Ct of the indicated housekeeping genes. The standard error is calculated from the standard deviations of individual primers:\n", "\n", "$$\n", "\\sigma_{housekeeping} = 0.5 * \\sqrt{\\sigma_{18S} + \\sigma_{Polr2a}} \\\\\n", "\\sigma_{\\Delta Ct} = 0.5 * \\sqrt{\\sigma_{housekeeping} + \\sigma_{gene \\: of \\: interest}}\n", "$$" ] }, { "cell_type": "markdown", "id": "70ac5858-b147-4714-a0d3-81ea964872c4", "metadata": {}, "source": [ "## ΔΔCt analysis" ] }, { "cell_type": "markdown", "id": "3be69720-5f3e-47e5-9042-1ca1f49ebd05", "metadata": {}, "source": [ "For ΔΔCt analysis, equipt requires an additional parameters to identify the experimental and control samples:" ] }, { "cell_type": "code", "execution_count": 6, "id": "bf12c1c6-678f-45af-8058-cac10324cf18", "metadata": { "tags": [] }, "outputs": [ { "data": { "text/html": [ "
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ExperimentalControlPrimerExp dCtddCtStdErrFoldChange
0Hdac3-1Scramble-1Ewsr13.7700000.0416670.0499440.971532
1Hdac3-1Scramble-1Fus3.7800000.0983330.0271830.934111
2Hdac3-1Scramble-1Hdac39.2100002.2183330.0808630.214889
3Hdac3-1Scramble-1Taf155.7966670.0283330.0464880.980552
4Hdac3-2Scramble-2Ewsr13.6466670.0933330.0541600.937354
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" ], "text/plain": [ " Experimental Control Primer Exp dCt ddCt StdErr FoldChange\n", "0 Hdac3-1 Scramble-1 Ewsr1 3.770000 0.041667 0.049944 0.971532\n", "1 Hdac3-1 Scramble-1 Fus 3.780000 0.098333 0.027183 0.934111\n", "2 Hdac3-1 Scramble-1 Hdac3 9.210000 2.218333 0.080863 0.214889\n", "3 Hdac3-1 Scramble-1 Taf15 5.796667 0.028333 0.046488 0.980552\n", "4 Hdac3-2 Scramble-2 Ewsr1 3.646667 0.093333 0.054160 0.937354" ] }, "execution_count": 6, "metadata": {}, "output_type": "execute_result" } ], "source": [ "housekeeping = ['18S','Polr2a']\n", "\n", "exp_ctrl = {'Hdac3-1':'Scramble-1',\n", " 'Hdac3-2':'Scramble-2',\n", " 'Fus-1':'Scramble-1',\n", " 'Fus-2':'Scramble-2',\n", " 'Taf15-1':'Scramble-1',\n", " 'Ewsr1-1':'Scramble-1',\n", " 'Ewsr1-2':'Scramble-2',\n", " 'Taf15-2':'Scramble-2'}\n", "\n", "ddct = equipt.deltact(df,\n", " housekeeping,\n", " reps,\n", " dilution=None,\n", " thresh=0.1,\n", " exp_ctrl=exp_ctrl,\n", " foldchange=True \n", " )\n", "\n", "ddct.head()" ] }, { "cell_type": "markdown", "id": "94e52ccd-02f2-44ad-9841-ba7f0f8dcda8", "metadata": {}, "source": [ "Equipt has performed the desired calculations. It also labels which experimental and control pairs it used so that we can easily check whether we made any errors in the exp_ctrl dictionary. Standard error for ΔΔCt is calculated from the ΔCt values:\n", "\n", "$$\n", "\\sigma_{\\Delta \\Delta Ct} = 0.5 * \\sqrt{\\sigma_{{\\Delta Ct}_{experimental}} + \\sigma_{{\\Delta Ct}_{experimental}}}\n", "$$\n", "\n", "Equipt does not return standard errors for fold change as the log-transformation makes these values difficult to meaningfully interpret. Examining the spread of fold changes among biological replicates is typically more meaningful than looking at the technical variation of an individual fold change (assuming that the error of the underlying ΔΔCt is acceptably low for the effect size). Because of these challenges, reporting the ΔΔCt values directly is often more honest to the data than reporting fold changes. Nonetheless, journals and peer reviewers generally expect to see fold changes, so equipt provides the option to output them automatically.\n", "\n", "Now that the desired values are available in a tidy dataframe, plotting the data with a high level data visualization package such as Holoviews is simple:" ] }, { "cell_type": "code", "execution_count": 7, "id": "c361e95f-67c5-4cc0-acb6-016278c0ae1c", "metadata": { "tags": [] }, "outputs": [ { "data": { "application/javascript": [ "(function(root) {\n", " function now() {\n", " return new Date();\n", " }\n", "\n", " var force = true;\n", " var py_version = '3.1.1'.replace('rc', '-rc.');\n", " var is_dev = py_version.indexOf(\"+\") !== -1 || py_version.indexOf(\"-\") !== -1;\n", " var reloading = false;\n", " var Bokeh = root.Bokeh;\n", " var bokeh_loaded = Bokeh != null && (Bokeh.version === py_version || (Bokeh.versions !== undefined && Bokeh.versions.has(py_version)));\n", "\n", " if (typeof (root._bokeh_timeout) === \"undefined\" || 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${name}\n", " window._bokeh_on_load()\n", " `\n", " document.head.appendChild(element);\n", " }\n", " if (!js_urls.length && !js_modules.length) {\n", " on_load()\n", " }\n", " };\n", "\n", " function inject_raw_css(css) {\n", " const element = document.createElement(\"style\");\n", " element.appendChild(document.createTextNode(css));\n", " document.body.appendChild(element);\n", " }\n", "\n", " var js_urls = [\"https://cdn.bokeh.org/bokeh/release/bokeh-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-gl-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-widgets-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-tables-3.1.1.min.js\", \"https://cdn.holoviz.org/panel/1.1.0/dist/panel.min.js\"];\n", " var js_modules = [];\n", " var js_exports = {};\n", " var css_urls = [];\n", " var inline_js = [ function(Bokeh) {\n", " Bokeh.set_log_level(\"info\");\n", " },\n", "function(Bokeh) {} // ensure no trailing comma for IE\n", " ];\n", "\n", " function run_inline_js() {\n", " if ((root.Bokeh !== undefined) || (force === true)) {\n", " for (var i = 0; i < inline_js.length; i++) {\n", " inline_js[i].call(root, root.Bokeh);\n", " }\n", " // Cache old bokeh versions\n", " if (Bokeh != undefined && !reloading) {\n", "\tvar NewBokeh = root.Bokeh;\n", "\tif (Bokeh.versions === undefined) {\n", "\t Bokeh.versions = new Map();\n", "\t}\n", "\tif (NewBokeh.version !== Bokeh.version) {\n", "\t Bokeh.versions.set(NewBokeh.version, NewBokeh)\n", "\t}\n", "\troot.Bokeh = Bokeh;\n", " }} else if (Date.now() < root._bokeh_timeout) {\n", " setTimeout(run_inline_js, 100);\n", " } else if (!root._bokeh_failed_load) {\n", " console.log(\"Bokeh: BokehJS failed to load within specified timeout.\");\n", " root._bokeh_failed_load = true;\n", " }\n", " root._bokeh_is_initializing = false\n", " }\n", "\n", " function load_or_wait() {\n", " // Implement a backoff loop that tries to ensure we do not load multiple\n", " // versions of Bokeh and its dependencies at the same time.\n", " // In recent versions we use the root._bokeh_is_initializing flag\n", " // to determine whether there is an ongoing attempt to initialize\n", " // bokeh, however for backward compatibility we also try to ensure\n", " // that we do not start loading a newer (Panel>=1.0 and Bokeh>3) version\n", " // before older versions are fully initialized.\n", " if (root._bokeh_is_initializing && Date.now() > root._bokeh_timeout) {\n", " root._bokeh_is_initializing = false;\n", " root._bokeh_onload_callbacks = undefined;\n", " console.log(\"Bokeh: BokehJS was loaded multiple times but one version failed to initialize.\");\n", " load_or_wait();\n", " } else if (root._bokeh_is_initializing || (typeof root._bokeh_is_initializing === \"undefined\" && root._bokeh_onload_callbacks !== undefined)) {\n", " setTimeout(load_or_wait, 100);\n", " } else {\n", " Bokeh = root.Bokeh;\n", " bokeh_loaded = Bokeh != null && (Bokeh.version === py_version || (Bokeh.versions !== undefined && Bokeh.versions.has(py_version)));\n", " root._bokeh_is_initializing = true\n", " root._bokeh_onload_callbacks = []\n", " if (!reloading && (!bokeh_loaded || is_dev)) {\n", "\troot.Bokeh = undefined;\n", " }\n", " load_libs(css_urls, js_urls, js_modules, js_exports, function() {\n", "\tconsole.debug(\"Bokeh: BokehJS plotting callback run at\", now());\n", "\trun_inline_js();\n", " });\n", " }\n", " }\n", " // Give older versions of the autoload script a head-start to ensure\n", " // they initialize before we start loading newer version.\n", " setTimeout(load_or_wait, 100)\n", "}(window));" ], "application/vnd.holoviews_load.v0+json": "(function(root) {\n function now() {\n return new Date();\n }\n\n var force = true;\n var py_version = '3.1.1'.replace('rc', '-rc.');\n var is_dev = py_version.indexOf(\"+\") !== -1 || py_version.indexOf(\"-\") !== -1;\n var reloading = false;\n var Bokeh = root.Bokeh;\n var bokeh_loaded = Bokeh != null && (Bokeh.version === py_version || (Bokeh.versions !== undefined && Bokeh.versions.has(py_version)));\n\n if (typeof (root._bokeh_timeout) === \"undefined\" || force) {\n root._bokeh_timeout = Date.now() + 5000;\n root._bokeh_failed_load = false;\n }\n\n function run_callbacks() {\n try {\n root._bokeh_onload_callbacks.forEach(function(callback) {\n if (callback != null)\n callback();\n });\n } finally {\n delete root._bokeh_onload_callbacks;\n }\n console.debug(\"Bokeh: all callbacks have finished\");\n }\n\n function load_libs(css_urls, js_urls, js_modules, js_exports, callback) {\n if (css_urls == null) css_urls = [];\n if (js_urls == null) js_urls = [];\n if (js_modules == null) js_modules = [];\n if (js_exports == null) js_exports = {};\n\n root._bokeh_onload_callbacks.push(callback);\n\n if (root._bokeh_is_loading > 0) {\n console.debug(\"Bokeh: BokehJS is being loaded, scheduling callback at\", now());\n return null;\n }\n if (js_urls.length === 0 && js_modules.length === 0 && Object.keys(js_exports).length === 0) {\n run_callbacks();\n return null;\n }\n if (!reloading) {\n console.debug(\"Bokeh: BokehJS not loaded, scheduling load and callback at\", now());\n }\n\n function on_load() {\n root._bokeh_is_loading--;\n if (root._bokeh_is_loading === 0) {\n console.debug(\"Bokeh: all BokehJS libraries/stylesheets loaded\");\n run_callbacks()\n }\n }\n window._bokeh_on_load = on_load\n\n function on_error() {\n console.error(\"failed to load \" + url);\n }\n\n var skip = [];\n if (window.requirejs) {\n window.requirejs.config({'packages': {}, 'paths': {'jspanel': 'https://cdn.jsdelivr.net/npm/jspanel4@4.12.0/dist/jspanel', 'jspanel-modal': 'https://cdn.jsdelivr.net/npm/jspanel4@4.12.0/dist/extensions/modal/jspanel.modal', 'jspanel-tooltip': 'https://cdn.jsdelivr.net/npm/jspanel4@4.12.0/dist/extensions/tooltip/jspanel.tooltip', 'jspanel-hint': 'https://cdn.jsdelivr.net/npm/jspanel4@4.12.0/dist/extensions/hint/jspanel.hint', 'jspanel-layout': 'https://cdn.jsdelivr.net/npm/jspanel4@4.12.0/dist/extensions/layout/jspanel.layout', 'jspanel-contextmenu': 'https://cdn.jsdelivr.net/npm/jspanel4@4.12.0/dist/extensions/contextmenu/jspanel.contextmenu', 'jspanel-dock': 'https://cdn.jsdelivr.net/npm/jspanel4@4.12.0/dist/extensions/dock/jspanel.dock', 'gridstack': 'https://cdn.jsdelivr.net/npm/gridstack@7.2.3/dist/gridstack-all', 'notyf': 'https://cdn.jsdelivr.net/npm/notyf@3/notyf.min'}, 'shim': {'jspanel': {'exports': 'jsPanel'}, 'gridstack': {'exports': 'GridStack'}}});\n require([\"jspanel\"], function(jsPanel) {\n\twindow.jsPanel = jsPanel\n\ton_load()\n })\n require([\"jspanel-modal\"], function() {\n\ton_load()\n })\n require([\"jspanel-tooltip\"], function() {\n\ton_load()\n })\n require([\"jspanel-hint\"], function() {\n\ton_load()\n })\n require([\"jspanel-layout\"], function() {\n\ton_load()\n })\n require([\"jspanel-contextmenu\"], function() {\n\ton_load()\n })\n require([\"jspanel-dock\"], function() {\n\ton_load()\n })\n require([\"gridstack\"], function(GridStack) {\n\twindow.GridStack = GridStack\n\ton_load()\n })\n require([\"notyf\"], function() {\n\ton_load()\n })\n root._bokeh_is_loading = css_urls.length + 9;\n } else {\n root._bokeh_is_loading = css_urls.length + js_urls.length + js_modules.length + Object.keys(js_exports).length;\n }\n\n var existing_stylesheets = []\n var links = document.getElementsByTagName('link')\n for (var i = 0; i < links.length; i++) {\n var link = links[i]\n if (link.href != null) {\n\texisting_stylesheets.push(link.href)\n }\n }\n for (var i = 0; i < css_urls.length; i++) {\n var url = css_urls[i];\n if (existing_stylesheets.indexOf(url) !== -1) {\n\ton_load()\n\tcontinue;\n }\n const element = document.createElement(\"link\");\n element.onload = on_load;\n element.onerror = on_error;\n element.rel = \"stylesheet\";\n element.type = \"text/css\";\n element.href = url;\n console.debug(\"Bokeh: injecting link tag for BokehJS stylesheet: \", url);\n document.body.appendChild(element);\n } if (((window['jsPanel'] !== undefined) && (!(window['jsPanel'] instanceof HTMLElement))) || window.requirejs) {\n var urls = ['https://cdn.holoviz.org/panel/1.1.0/dist/bundled/floatpanel/jspanel4@4.12.0/dist/jspanel.js', 'https://cdn.holoviz.org/panel/1.1.0/dist/bundled/floatpanel/jspanel4@4.12.0/dist/extensions/modal/jspanel.modal.js', 'https://cdn.holoviz.org/panel/1.1.0/dist/bundled/floatpanel/jspanel4@4.12.0/dist/extensions/tooltip/jspanel.tooltip.js', 'https://cdn.holoviz.org/panel/1.1.0/dist/bundled/floatpanel/jspanel4@4.12.0/dist/extensions/hint/jspanel.hint.js', 'https://cdn.holoviz.org/panel/1.1.0/dist/bundled/floatpanel/jspanel4@4.12.0/dist/extensions/layout/jspanel.layout.js', 'https://cdn.holoviz.org/panel/1.1.0/dist/bundled/floatpanel/jspanel4@4.12.0/dist/extensions/contextmenu/jspanel.contextmenu.js', 'https://cdn.holoviz.org/panel/1.1.0/dist/bundled/floatpanel/jspanel4@4.12.0/dist/extensions/dock/jspanel.dock.js'];\n for (var i = 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!== -1) {\n\tif (!window.requirejs) {\n\t on_load();\n\t}\n\tcontinue;\n }\n var element = document.createElement('script');\n element.onload = on_load;\n element.onerror = on_error;\n element.async = false;\n element.src = url;\n console.debug(\"Bokeh: injecting script tag for BokehJS library: \", url);\n document.head.appendChild(element);\n }\n for (var i = 0; i < js_modules.length; i++) {\n var url = js_modules[i];\n if (skip.indexOf(url) !== -1 || existing_scripts.indexOf(url) !== -1) {\n\tif (!window.requirejs) {\n\t on_load();\n\t}\n\tcontinue;\n }\n var element = document.createElement('script');\n element.onload = on_load;\n element.onerror = on_error;\n element.async = false;\n element.src = url;\n element.type = \"module\";\n console.debug(\"Bokeh: injecting script tag for BokehJS library: \", url);\n document.head.appendChild(element);\n }\n for (const name in js_exports) {\n var url = js_exports[name];\n if (skip.indexOf(url) >= 0 || root[name] != null) {\n\tif 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});\n", " };\n", " var comm = {\n", " send: sendClosure\n", " };\n", " }\n", " window.PyViz.comms[comm_id] = comm;\n", " return comm;\n", " }\n", " window.PyViz.comm_manager = new JupyterCommManager();\n", " \n", "\n", "\n", "var JS_MIME_TYPE = 'application/javascript';\n", "var HTML_MIME_TYPE = 'text/html';\n", "var EXEC_MIME_TYPE = 'application/vnd.holoviews_exec.v0+json';\n", "var CLASS_NAME = 'output';\n", "\n", "/**\n", " * Render data to the DOM node\n", " */\n", "function render(props, node) {\n", " var div = document.createElement(\"div\");\n", " var script = document.createElement(\"script\");\n", " node.appendChild(div);\n", " node.appendChild(script);\n", "}\n", "\n", "/**\n", " * Handle when a new output is added\n", " */\n", "function handle_add_output(event, handle) {\n", " var output_area = handle.output_area;\n", " var output = handle.output;\n", " if ((output.data == undefined) || (!output.data.hasOwnProperty(EXEC_MIME_TYPE))) {\n", " return\n", " }\n", " var id = output.metadata[EXEC_MIME_TYPE][\"id\"];\n", " var toinsert = output_area.element.find(\".\" + CLASS_NAME.split(' ')[0]);\n", " if (id !== undefined) {\n", " var nchildren = toinsert.length;\n", " var html_node = toinsert[nchildren-1].children[0];\n", " html_node.innerHTML = output.data[HTML_MIME_TYPE];\n", " var scripts = [];\n", " var nodelist = html_node.querySelectorAll(\"script\");\n", " for (var i in nodelist) {\n", " if (nodelist.hasOwnProperty(i)) {\n", " scripts.push(nodelist[i])\n", " }\n", " }\n", "\n", " scripts.forEach( function (oldScript) {\n", " var newScript = document.createElement(\"script\");\n", " var attrs = [];\n", " var nodemap = oldScript.attributes;\n", " for (var j in nodemap) {\n", " if (nodemap.hasOwnProperty(j)) {\n", " attrs.push(nodemap[j])\n", " }\n", " }\n", " attrs.forEach(function(attr) { newScript.setAttribute(attr.name, attr.value) });\n", " newScript.appendChild(document.createTextNode(oldScript.innerHTML));\n", " oldScript.parentNode.replaceChild(newScript, oldScript);\n", " });\n", " if (JS_MIME_TYPE in output.data) {\n", " toinsert[nchildren-1].children[1].textContent = output.data[JS_MIME_TYPE];\n", " }\n", " output_area._hv_plot_id = id;\n", " if ((window.Bokeh !== undefined) && (id in Bokeh.index)) {\n", " window.PyViz.plot_index[id] = Bokeh.index[id];\n", " } else {\n", " window.PyViz.plot_index[id] = null;\n", " }\n", " } else if (output.metadata[EXEC_MIME_TYPE][\"server_id\"] !== undefined) {\n", " var bk_div = document.createElement(\"div\");\n", " bk_div.innerHTML = output.data[HTML_MIME_TYPE];\n", " var script_attrs = bk_div.children[0].attributes;\n", " for (var i = 0; i < script_attrs.length; i++) {\n", " toinsert[toinsert.length - 1].childNodes[1].setAttribute(script_attrs[i].name, script_attrs[i].value);\n", " }\n", " // store reference to server id on output_area\n", " output_area._bokeh_server_id = output.metadata[EXEC_MIME_TYPE][\"server_id\"];\n", " }\n", "}\n", "\n", "/**\n", " * Handle when an output is cleared or removed\n", " */\n", "function handle_clear_output(event, handle) {\n", " var id = handle.cell.output_area._hv_plot_id;\n", " var server_id = handle.cell.output_area._bokeh_server_id;\n", " if (((id === undefined) || !(id in PyViz.plot_index)) && (server_id !== undefined)) { return; }\n", " var comm = window.PyViz.comm_manager.get_client_comm(\"hv-extension-comm\", \"hv-extension-comm\", function () {});\n", " if (server_id !== null) {\n", " comm.send({event_type: 'server_delete', 'id': server_id});\n", " return;\n", " } else if (comm !== null) {\n", " comm.send({event_type: 'delete', 'id': id});\n", " }\n", " delete PyViz.plot_index[id];\n", " if ((window.Bokeh !== undefined) & (id in window.Bokeh.index)) {\n", " var doc = window.Bokeh.index[id].model.document\n", " doc.clear();\n", " const i = window.Bokeh.documents.indexOf(doc);\n", " if (i > -1) {\n", " window.Bokeh.documents.splice(i, 1);\n", " }\n", " }\n", "}\n", "\n", "/**\n", " * Handle kernel restart event\n", " */\n", "function handle_kernel_cleanup(event, handle) {\n", " delete PyViz.comms[\"hv-extension-comm\"];\n", " window.PyViz.plot_index = {}\n", "}\n", "\n", "/**\n", " * Handle update_display_data messages\n", " */\n", "function handle_update_output(event, handle) {\n", " handle_clear_output(event, {cell: {output_area: handle.output_area}})\n", " handle_add_output(event, handle)\n", "}\n", "\n", "function register_renderer(events, OutputArea) {\n", " function append_mime(data, metadata, element) {\n", " // create a DOM node to render to\n", " var toinsert = this.create_output_subarea(\n", " metadata,\n", " CLASS_NAME,\n", " EXEC_MIME_TYPE\n", " );\n", " this.keyboard_manager.register_events(toinsert);\n", " // Render to node\n", " var props = {data: data, metadata: metadata[EXEC_MIME_TYPE]};\n", " render(props, toinsert[0]);\n", " element.append(toinsert);\n", " return toinsert\n", " }\n", "\n", " events.on('output_added.OutputArea', handle_add_output);\n", " events.on('output_updated.OutputArea', handle_update_output);\n", " events.on('clear_output.CodeCell', handle_clear_output);\n", " events.on('delete.Cell', handle_clear_output);\n", " events.on('kernel_ready.Kernel', handle_kernel_cleanup);\n", "\n", " OutputArea.prototype.register_mime_type(EXEC_MIME_TYPE, append_mime, {\n", " safe: true,\n", " index: 0\n", " });\n", "}\n", "\n", "if (window.Jupyter !== undefined) {\n", " try {\n", " var events = require('base/js/events');\n", " var OutputArea = require('notebook/js/outputarea').OutputArea;\n", " if (OutputArea.prototype.mime_types().indexOf(EXEC_MIME_TYPE) == -1) {\n", " register_renderer(events, OutputArea);\n", " }\n", " } catch(err) {\n", " }\n", "}\n" ], "application/vnd.holoviews_load.v0+json": "\nif ((window.PyViz === undefined) || (window.PyViz instanceof HTMLElement)) {\n window.PyViz = {comms: {}, comm_status:{}, kernels:{}, receivers: {}, plot_index: []}\n}\n\n\n function JupyterCommManager() {\n }\n\n JupyterCommManager.prototype.register_target = function(plot_id, comm_id, msg_handler) {\n if (window.comm_manager || ((window.Jupyter !== undefined) && (Jupyter.notebook.kernel != null))) {\n var comm_manager = window.comm_manager || Jupyter.notebook.kernel.comm_manager;\n comm_manager.register_target(comm_id, function(comm) {\n comm.on_msg(msg_handler);\n });\n } else if ((plot_id in window.PyViz.kernels) && (window.PyViz.kernels[plot_id])) {\n window.PyViz.kernels[plot_id].registerCommTarget(comm_id, function(comm) {\n comm.onMsg = msg_handler;\n });\n } else if (typeof google != 'undefined' && google.colab.kernel != null) {\n google.colab.kernel.comms.registerTarget(comm_id, (comm) => {\n var messages = comm.messages[Symbol.asyncIterator]();\n function processIteratorResult(result) {\n var message = result.value;\n console.log(message)\n var content = {data: message.data, comm_id};\n var buffers = []\n for (var buffer of message.buffers || []) {\n buffers.push(new DataView(buffer))\n }\n var metadata = message.metadata || {};\n var msg = {content, buffers, metadata}\n msg_handler(msg);\n return messages.next().then(processIteratorResult);\n }\n return messages.next().then(processIteratorResult);\n })\n }\n }\n\n JupyterCommManager.prototype.get_client_comm = function(plot_id, comm_id, msg_handler) {\n if (comm_id in window.PyViz.comms) {\n return window.PyViz.comms[comm_id];\n } else if (window.comm_manager || ((window.Jupyter !== undefined) && (Jupyter.notebook.kernel != null))) {\n var comm_manager = window.comm_manager || Jupyter.notebook.kernel.comm_manager;\n var comm = comm_manager.new_comm(comm_id, {}, {}, {}, comm_id);\n if (msg_handler) {\n comm.on_msg(msg_handler);\n }\n } else if ((plot_id in window.PyViz.kernels) && (window.PyViz.kernels[plot_id])) {\n var comm = window.PyViz.kernels[plot_id].connectToComm(comm_id);\n comm.open();\n if (msg_handler) {\n comm.onMsg = msg_handler;\n }\n } else if (typeof google != 'undefined' && google.colab.kernel != null) {\n var comm_promise = google.colab.kernel.comms.open(comm_id)\n comm_promise.then((comm) => {\n window.PyViz.comms[comm_id] = comm;\n if (msg_handler) {\n var messages = comm.messages[Symbol.asyncIterator]();\n function processIteratorResult(result) {\n var message = result.value;\n var content = {data: message.data};\n var metadata = message.metadata || {comm_id};\n var msg = {content, metadata}\n msg_handler(msg);\n return messages.next().then(processIteratorResult);\n }\n return messages.next().then(processIteratorResult);\n }\n }) \n var sendClosure = (data, metadata, buffers, disposeOnDone) => {\n return comm_promise.then((comm) => {\n comm.send(data, metadata, buffers, disposeOnDone);\n });\n };\n var comm = {\n send: sendClosure\n };\n }\n window.PyViz.comms[comm_id] = comm;\n return comm;\n }\n window.PyViz.comm_manager = new JupyterCommManager();\n \n\n\nvar JS_MIME_TYPE = 'application/javascript';\nvar HTML_MIME_TYPE = 'text/html';\nvar EXEC_MIME_TYPE = 'application/vnd.holoviews_exec.v0+json';\nvar CLASS_NAME = 'output';\n\n/**\n * Render data to the DOM node\n */\nfunction render(props, node) {\n var div = document.createElement(\"div\");\n var script = document.createElement(\"script\");\n node.appendChild(div);\n node.appendChild(script);\n}\n\n/**\n * Handle when a new output is added\n */\nfunction handle_add_output(event, handle) {\n var output_area = handle.output_area;\n var output = handle.output;\n if ((output.data == undefined) || (!output.data.hasOwnProperty(EXEC_MIME_TYPE))) {\n return\n }\n var id = output.metadata[EXEC_MIME_TYPE][\"id\"];\n var toinsert = output_area.element.find(\".\" + CLASS_NAME.split(' ')[0]);\n if (id !== undefined) {\n var nchildren = toinsert.length;\n var html_node = toinsert[nchildren-1].children[0];\n html_node.innerHTML = output.data[HTML_MIME_TYPE];\n var scripts = [];\n var nodelist = html_node.querySelectorAll(\"script\");\n for (var i in nodelist) {\n if (nodelist.hasOwnProperty(i)) {\n scripts.push(nodelist[i])\n }\n }\n\n scripts.forEach( function (oldScript) {\n var newScript = document.createElement(\"script\");\n var attrs = [];\n var nodemap = oldScript.attributes;\n for (var j in nodemap) {\n if (nodemap.hasOwnProperty(j)) {\n attrs.push(nodemap[j])\n }\n }\n attrs.forEach(function(attr) { newScript.setAttribute(attr.name, attr.value) });\n newScript.appendChild(document.createTextNode(oldScript.innerHTML));\n oldScript.parentNode.replaceChild(newScript, oldScript);\n });\n if (JS_MIME_TYPE in output.data) {\n toinsert[nchildren-1].children[1].textContent = output.data[JS_MIME_TYPE];\n }\n output_area._hv_plot_id = id;\n if ((window.Bokeh !== undefined) && (id in Bokeh.index)) {\n window.PyViz.plot_index[id] = Bokeh.index[id];\n } else {\n window.PyViz.plot_index[id] = null;\n }\n } else if (output.metadata[EXEC_MIME_TYPE][\"server_id\"] !== undefined) {\n var bk_div = document.createElement(\"div\");\n bk_div.innerHTML = output.data[HTML_MIME_TYPE];\n var script_attrs = bk_div.children[0].attributes;\n for (var i = 0; i < script_attrs.length; i++) {\n toinsert[toinsert.length - 1].childNodes[1].setAttribute(script_attrs[i].name, script_attrs[i].value);\n }\n // store reference to server id on output_area\n output_area._bokeh_server_id = output.metadata[EXEC_MIME_TYPE][\"server_id\"];\n }\n}\n\n/**\n * Handle when an output is cleared or removed\n */\nfunction handle_clear_output(event, handle) {\n var id = handle.cell.output_area._hv_plot_id;\n var server_id = handle.cell.output_area._bokeh_server_id;\n if (((id === undefined) || !(id in PyViz.plot_index)) && (server_id !== undefined)) { return; }\n var comm = window.PyViz.comm_manager.get_client_comm(\"hv-extension-comm\", \"hv-extension-comm\", function () {});\n if (server_id !== null) {\n comm.send({event_type: 'server_delete', 'id': server_id});\n return;\n } else if (comm !== null) {\n comm.send({event_type: 'delete', 'id': id});\n }\n delete PyViz.plot_index[id];\n if ((window.Bokeh !== undefined) & (id in window.Bokeh.index)) {\n var doc = window.Bokeh.index[id].model.document\n doc.clear();\n const i = window.Bokeh.documents.indexOf(doc);\n if (i > -1) {\n window.Bokeh.documents.splice(i, 1);\n }\n }\n}\n\n/**\n * Handle kernel restart event\n */\nfunction handle_kernel_cleanup(event, handle) {\n delete PyViz.comms[\"hv-extension-comm\"];\n window.PyViz.plot_index = {}\n}\n\n/**\n * Handle update_display_data messages\n */\nfunction handle_update_output(event, handle) {\n handle_clear_output(event, {cell: {output_area: handle.output_area}})\n handle_add_output(event, handle)\n}\n\nfunction register_renderer(events, OutputArea) {\n function append_mime(data, metadata, element) {\n // create a DOM node to render to\n var toinsert = this.create_output_subarea(\n metadata,\n CLASS_NAME,\n EXEC_MIME_TYPE\n );\n this.keyboard_manager.register_events(toinsert);\n // Render to node\n var props = {data: data, metadata: metadata[EXEC_MIME_TYPE]};\n render(props, toinsert[0]);\n element.append(toinsert);\n return toinsert\n }\n\n events.on('output_added.OutputArea', handle_add_output);\n events.on('output_updated.OutputArea', handle_update_output);\n events.on('clear_output.CodeCell', handle_clear_output);\n events.on('delete.Cell', handle_clear_output);\n events.on('kernel_ready.Kernel', handle_kernel_cleanup);\n\n OutputArea.prototype.register_mime_type(EXEC_MIME_TYPE, append_mime, {\n safe: true,\n index: 0\n });\n}\n\nif (window.Jupyter !== undefined) {\n try {\n var events = require('base/js/events');\n var OutputArea = require('notebook/js/outputarea').OutputArea;\n if (OutputArea.prototype.mime_types().indexOf(EXEC_MIME_TYPE) == -1) {\n register_renderer(events, OutputArea);\n }\n } catch(err) {\n }\n}\n" }, "metadata": {}, "output_type": "display_data" }, { "data": { "text/html": [ "" ] }, "metadata": {}, "output_type": "display_data" }, { "data": { "text/html": [ "\n", "
\n", "\n", "\n", "\n", " \n", " \n", "\n", "\n", "\n", "\n", "
\n" ] }, "metadata": {}, "output_type": "display_data" }, { "data": { "text/html": [ "\n", "
\n", " \n", " Loading BokehJS ...\n", "
\n" ] }, "metadata": {}, "output_type": "display_data" }, { "data": { "application/javascript": [ "(function(root) {\n", " function now() {\n", " return new Date();\n", " }\n", "\n", " const force = true;\n", "\n", " if (typeof root._bokeh_onload_callbacks === \"undefined\" || force === true) {\n", " root._bokeh_onload_callbacks = [];\n", " root._bokeh_is_loading = undefined;\n", " }\n", "\n", "const JS_MIME_TYPE = 'application/javascript';\n", " const HTML_MIME_TYPE = 'text/html';\n", " const EXEC_MIME_TYPE = 'application/vnd.bokehjs_exec.v0+json';\n", " const CLASS_NAME = 'output_bokeh rendered_html';\n", "\n", " /**\n", " * Render data to the DOM node\n", " */\n", " function render(props, node) {\n", " const script = document.createElement(\"script\");\n", " node.appendChild(script);\n", " }\n", "\n", " /**\n", " * Handle when an output is cleared or removed\n", " */\n", " function handleClearOutput(event, handle) {\n", " const cell = handle.cell;\n", "\n", " const id = cell.output_area._bokeh_element_id;\n", " const server_id = cell.output_area._bokeh_server_id;\n", " // Clean up Bokeh references\n", " if (id != null && id in Bokeh.index) {\n", " Bokeh.index[id].model.document.clear();\n", " delete Bokeh.index[id];\n", " }\n", "\n", " if (server_id !== undefined) {\n", " // Clean up Bokeh references\n", " const cmd_clean = \"from bokeh.io.state import curstate; print(curstate().uuid_to_server['\" + server_id + \"'].get_sessions()[0].document.roots[0]._id)\";\n", " cell.notebook.kernel.execute(cmd_clean, {\n", " iopub: {\n", " output: function(msg) {\n", " const id = msg.content.text.trim();\n", " if (id in Bokeh.index) {\n", " Bokeh.index[id].model.document.clear();\n", " delete Bokeh.index[id];\n", " }\n", " }\n", " }\n", " });\n", " // Destroy server and session\n", " const cmd_destroy = \"import bokeh.io.notebook as ion; ion.destroy_server('\" + server_id + \"')\";\n", " cell.notebook.kernel.execute(cmd_destroy);\n", " }\n", " }\n", "\n", " /**\n", " * Handle when a new output is added\n", " */\n", " function handleAddOutput(event, handle) {\n", " const output_area = handle.output_area;\n", " const output = handle.output;\n", "\n", " // limit handleAddOutput to display_data with EXEC_MIME_TYPE content only\n", " if ((output.output_type != \"display_data\") || (!Object.prototype.hasOwnProperty.call(output.data, EXEC_MIME_TYPE))) {\n", " return\n", " }\n", "\n", " const toinsert = output_area.element.find(\".\" + CLASS_NAME.split(' ')[0]);\n", "\n", " if (output.metadata[EXEC_MIME_TYPE][\"id\"] !== undefined) {\n", " toinsert[toinsert.length - 1].firstChild.textContent = output.data[JS_MIME_TYPE];\n", " // store reference to embed id on output_area\n", " output_area._bokeh_element_id = output.metadata[EXEC_MIME_TYPE][\"id\"];\n", " }\n", " if (output.metadata[EXEC_MIME_TYPE][\"server_id\"] !== undefined) {\n", " const bk_div = document.createElement(\"div\");\n", " bk_div.innerHTML = output.data[HTML_MIME_TYPE];\n", " const script_attrs = bk_div.children[0].attributes;\n", " for (let i = 0; i < script_attrs.length; i++) {\n", " toinsert[toinsert.length - 1].firstChild.setAttribute(script_attrs[i].name, script_attrs[i].value);\n", " toinsert[toinsert.length - 1].firstChild.textContent = bk_div.children[0].textContent\n", " }\n", " // store reference to server id on output_area\n", " output_area._bokeh_server_id = output.metadata[EXEC_MIME_TYPE][\"server_id\"];\n", " }\n", " }\n", "\n", " function register_renderer(events, OutputArea) {\n", "\n", " function append_mime(data, metadata, element) {\n", " // create a DOM node to render to\n", " const toinsert = this.create_output_subarea(\n", " metadata,\n", " CLASS_NAME,\n", " EXEC_MIME_TYPE\n", " );\n", " this.keyboard_manager.register_events(toinsert);\n", " // Render to node\n", " const props = {data: data, metadata: metadata[EXEC_MIME_TYPE]};\n", " render(props, toinsert[toinsert.length - 1]);\n", " element.append(toinsert);\n", " return toinsert\n", " }\n", "\n", " /* Handle when an output is cleared or removed */\n", " events.on('clear_output.CodeCell', handleClearOutput);\n", " events.on('delete.Cell', handleClearOutput);\n", "\n", " /* Handle when a new output is added */\n", " events.on('output_added.OutputArea', handleAddOutput);\n", "\n", " /**\n", " * Register the mime type and append_mime function with output_area\n", " */\n", " OutputArea.prototype.register_mime_type(EXEC_MIME_TYPE, append_mime, {\n", " /* Is output safe? */\n", " safe: true,\n", " /* Index of renderer in `output_area.display_order` */\n", " index: 0\n", " });\n", " }\n", "\n", " // register the mime type if in Jupyter Notebook environment and previously unregistered\n", " if (root.Jupyter !== undefined) {\n", " const events = require('base/js/events');\n", " const OutputArea = require('notebook/js/outputarea').OutputArea;\n", "\n", " if (OutputArea.prototype.mime_types().indexOf(EXEC_MIME_TYPE) == -1) {\n", " register_renderer(events, OutputArea);\n", " }\n", " }\n", " if (typeof (root._bokeh_timeout) === \"undefined\" || force === true) {\n", " root._bokeh_timeout = Date.now() + 5000;\n", " root._bokeh_failed_load = false;\n", " }\n", "\n", " const NB_LOAD_WARNING = {'data': {'text/html':\n", " \"
\\n\"+\n", " \"

\\n\"+\n", " \"BokehJS does not appear to have successfully loaded. If loading BokehJS from CDN, this \\n\"+\n", " \"may be due to a slow or bad network connection. Possible fixes:\\n\"+\n", " \"

\\n\"+\n", " \"\\n\"+\n", " \"\\n\"+\n", " \"from bokeh.resources import INLINE\\n\"+\n", " \"output_notebook(resources=INLINE)\\n\"+\n", " \"\\n\"+\n", " \"
\"}};\n", "\n", " function display_loaded() {\n", " const el = document.getElementById(\"db6b6f89-83fc-4e37-8317-f2ca17e96791\");\n", " if (el != null) {\n", " el.textContent = \"BokehJS is loading...\";\n", " }\n", " if (root.Bokeh !== undefined) {\n", " if (el != null) {\n", " el.textContent = \"BokehJS \" + root.Bokeh.version + \" successfully loaded.\";\n", " }\n", " } else if (Date.now() < root._bokeh_timeout) {\n", " setTimeout(display_loaded, 100)\n", " }\n", " }\n", "\n", " function run_callbacks() {\n", " try {\n", " root._bokeh_onload_callbacks.forEach(function(callback) {\n", " if (callback != null)\n", " callback();\n", " });\n", " } finally {\n", " delete root._bokeh_onload_callbacks\n", " }\n", " console.debug(\"Bokeh: all callbacks have finished\");\n", " }\n", "\n", " function load_libs(css_urls, js_urls, callback) {\n", " if (css_urls == null) css_urls = [];\n", " if (js_urls == null) js_urls = [];\n", "\n", " root._bokeh_onload_callbacks.push(callback);\n", " if (root._bokeh_is_loading > 0) {\n", " console.debug(\"Bokeh: BokehJS is being loaded, scheduling callback at\", now());\n", " return null;\n", " }\n", " if (js_urls == null || js_urls.length === 0) {\n", " run_callbacks();\n", " return null;\n", " }\n", " console.debug(\"Bokeh: BokehJS not loaded, scheduling load and callback at\", now());\n", " root._bokeh_is_loading = css_urls.length + js_urls.length;\n", "\n", " function on_load() {\n", " root._bokeh_is_loading--;\n", " if (root._bokeh_is_loading === 0) {\n", " console.debug(\"Bokeh: all BokehJS libraries/stylesheets loaded\");\n", " run_callbacks()\n", " }\n", " }\n", "\n", " function on_error(url) {\n", " console.error(\"failed to load \" + url);\n", " }\n", "\n", " for (let i = 0; i < css_urls.length; i++) {\n", " const url = css_urls[i];\n", " const element = document.createElement(\"link\");\n", " element.onload = on_load;\n", " element.onerror = on_error.bind(null, url);\n", " element.rel = \"stylesheet\";\n", " element.type = \"text/css\";\n", " element.href = url;\n", " console.debug(\"Bokeh: injecting link tag for BokehJS stylesheet: \", url);\n", " document.body.appendChild(element);\n", " }\n", "\n", " for (let i = 0; i < js_urls.length; i++) {\n", " const url = js_urls[i];\n", " const element = document.createElement('script');\n", " element.onload = on_load;\n", " element.onerror = on_error.bind(null, url);\n", " element.async = false;\n", " element.src = url;\n", " console.debug(\"Bokeh: injecting script tag for BokehJS library: \", url);\n", " document.head.appendChild(element);\n", " }\n", " };\n", "\n", " function inject_raw_css(css) {\n", " const element = document.createElement(\"style\");\n", " element.appendChild(document.createTextNode(css));\n", " document.body.appendChild(element);\n", " }\n", "\n", " const js_urls = [\"https://cdn.bokeh.org/bokeh/release/bokeh-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-gl-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-widgets-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-tables-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-mathjax-3.1.1.min.js\", \"https://unpkg.com/@holoviz/panel@1.1.0/dist/panel.min.js\"];\n", " const css_urls = [];\n", "\n", " const inline_js = [ function(Bokeh) {\n", " Bokeh.set_log_level(\"info\");\n", " },\n", "function(Bokeh) {\n", " }\n", " ];\n", "\n", " function run_inline_js() {\n", " if (root.Bokeh !== undefined || force === true) {\n", " for (let i = 0; i < inline_js.length; i++) {\n", " inline_js[i].call(root, root.Bokeh);\n", " }\n", "if (force === true) {\n", " display_loaded();\n", " }} else if (Date.now() < root._bokeh_timeout) {\n", " setTimeout(run_inline_js, 100);\n", " } else if (!root._bokeh_failed_load) {\n", " console.log(\"Bokeh: BokehJS failed to load within specified timeout.\");\n", " root._bokeh_failed_load = true;\n", " } else if (force !== true) {\n", " const cell = $(document.getElementById(\"db6b6f89-83fc-4e37-8317-f2ca17e96791\")).parents('.cell').data().cell;\n", " cell.output_area.append_execute_result(NB_LOAD_WARNING)\n", " }\n", " }\n", "\n", " if (root._bokeh_is_loading === 0) {\n", " console.debug(\"Bokeh: BokehJS loaded, going straight to plotting\");\n", " run_inline_js();\n", " } else {\n", " load_libs(css_urls, js_urls, function() {\n", " console.debug(\"Bokeh: BokehJS plotting callback run at\", now());\n", " run_inline_js();\n", " });\n", " }\n", "}(window));" ], "application/vnd.bokehjs_load.v0+json": "(function(root) {\n function now() {\n return new Date();\n }\n\n const force = true;\n\n if (typeof root._bokeh_onload_callbacks === \"undefined\" || force === true) {\n root._bokeh_onload_callbacks = [];\n root._bokeh_is_loading = undefined;\n }\n\n\n if (typeof (root._bokeh_timeout) === \"undefined\" || force === true) {\n root._bokeh_timeout = Date.now() + 5000;\n root._bokeh_failed_load = false;\n }\n\n const NB_LOAD_WARNING = {'data': {'text/html':\n \"
\\n\"+\n \"

\\n\"+\n \"BokehJS does not appear to have successfully loaded. If loading BokehJS from CDN, this \\n\"+\n \"may be due to a slow or bad network connection. Possible fixes:\\n\"+\n \"

\\n\"+\n \"\\n\"+\n \"\\n\"+\n \"from bokeh.resources import INLINE\\n\"+\n \"output_notebook(resources=INLINE)\\n\"+\n \"\\n\"+\n \"
\"}};\n\n function display_loaded() {\n const el = document.getElementById(\"db6b6f89-83fc-4e37-8317-f2ca17e96791\");\n if (el != null) {\n el.textContent = \"BokehJS is loading...\";\n }\n if (root.Bokeh !== undefined) {\n if (el != null) {\n el.textContent = \"BokehJS \" + root.Bokeh.version + \" successfully loaded.\";\n }\n } else if (Date.now() < root._bokeh_timeout) {\n setTimeout(display_loaded, 100)\n }\n }\n\n function run_callbacks() {\n try {\n root._bokeh_onload_callbacks.forEach(function(callback) {\n if (callback != null)\n callback();\n });\n } finally {\n delete root._bokeh_onload_callbacks\n }\n console.debug(\"Bokeh: all callbacks have finished\");\n }\n\n function load_libs(css_urls, js_urls, callback) {\n if (css_urls == null) css_urls = [];\n if (js_urls == null) js_urls = [];\n\n root._bokeh_onload_callbacks.push(callback);\n if (root._bokeh_is_loading > 0) {\n console.debug(\"Bokeh: BokehJS is being loaded, scheduling callback at\", now());\n return null;\n }\n if (js_urls == null || js_urls.length === 0) {\n run_callbacks();\n return null;\n }\n console.debug(\"Bokeh: BokehJS not loaded, scheduling load and callback at\", now());\n root._bokeh_is_loading = css_urls.length + js_urls.length;\n\n function on_load() {\n root._bokeh_is_loading--;\n if (root._bokeh_is_loading === 0) {\n console.debug(\"Bokeh: all BokehJS libraries/stylesheets loaded\");\n run_callbacks()\n }\n }\n\n function on_error(url) {\n console.error(\"failed to load \" + url);\n }\n\n for (let i = 0; i < css_urls.length; i++) {\n const url = css_urls[i];\n const element = document.createElement(\"link\");\n element.onload = on_load;\n element.onerror = on_error.bind(null, url);\n element.rel = \"stylesheet\";\n element.type = \"text/css\";\n element.href = url;\n console.debug(\"Bokeh: injecting link tag for BokehJS stylesheet: \", url);\n document.body.appendChild(element);\n }\n\n for (let i = 0; i < js_urls.length; i++) {\n const url = js_urls[i];\n const element = document.createElement('script');\n element.onload = on_load;\n element.onerror = on_error.bind(null, url);\n element.async = false;\n element.src = url;\n console.debug(\"Bokeh: injecting script tag for BokehJS library: \", url);\n document.head.appendChild(element);\n }\n };\n\n function inject_raw_css(css) {\n const element = document.createElement(\"style\");\n element.appendChild(document.createTextNode(css));\n document.body.appendChild(element);\n }\n\n const js_urls = [\"https://cdn.bokeh.org/bokeh/release/bokeh-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-gl-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-widgets-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-tables-3.1.1.min.js\", \"https://cdn.bokeh.org/bokeh/release/bokeh-mathjax-3.1.1.min.js\", \"https://unpkg.com/@holoviz/panel@1.1.0/dist/panel.min.js\"];\n const css_urls = [];\n\n const inline_js = [ function(Bokeh) {\n Bokeh.set_log_level(\"info\");\n },\nfunction(Bokeh) {\n }\n ];\n\n function run_inline_js() {\n if (root.Bokeh !== undefined || force === true) {\n for (let i = 0; i < inline_js.length; i++) {\n inline_js[i].call(root, root.Bokeh);\n }\nif (force === true) {\n display_loaded();\n }} else if (Date.now() < root._bokeh_timeout) {\n setTimeout(run_inline_js, 100);\n } else if (!root._bokeh_failed_load) {\n console.log(\"Bokeh: BokehJS failed to load within specified timeout.\");\n root._bokeh_failed_load = true;\n } else if (force !== true) {\n const cell = $(document.getElementById(\"db6b6f89-83fc-4e37-8317-f2ca17e96791\")).parents('.cell').data().cell;\n cell.output_area.append_execute_result(NB_LOAD_WARNING)\n }\n }\n\n if (root._bokeh_is_loading === 0) {\n console.debug(\"Bokeh: BokehJS loaded, going straight to plotting\");\n run_inline_js();\n } else {\n load_libs(css_urls, js_urls, function() {\n console.debug(\"Bokeh: BokehJS plotting callback run at\", now());\n run_inline_js();\n });\n }\n}(window));" }, "metadata": {}, "output_type": "display_data" } ], "source": [ "# Import plotting packages\n", "import holoviews as hv\n", "import bokeh.io\n", "\n", "hv.extension('bokeh')\n", "bokeh.io.output_notebook()" ] }, { "cell_type": "code", "execution_count": 8, "id": "239fbb97-87b5-400a-9562-c98fb5fe632a", "metadata": { "tags": [] }, "outputs": [ { "data": {}, "metadata": {}, "output_type": "display_data" }, { "data": { "application/vnd.holoviews_exec.v0+json": "", "text/html": [ "
\n", "
\n", "
\n", "" ], "text/plain": [ ":BoxWhisker [Experimental,Primer] (FoldChange)" ] }, "execution_count": 8, "metadata": { "application/vnd.holoviews_exec.v0+json": { "id": "p1002" } }, "output_type": "execute_result" } ], "source": [ "# Remove replicate identifier\n", "ddct['Experimental'] = [i.split('-')[0] for i in ddct['Experimental']]\n", "\n", "# Plot\n", "boxwhisker = hv.BoxWhisker(data=ddct,\n", " kdims=['Experimental', 'Primer'],\n", " vdims=['FoldChange'],\n", " ).opts(width=600,\n", " xrotation=45,\n", " box_color='Primer',\n", " cmap={'Fus':'#F58817',\n", " 'Ewsr1':'#1B76B7',\n", " 'Taf15':'#BD7DAC',\n", " 'Hdac3':'#4AA177'}\n", " )\n", "\n", "boxwhisker" ] }, { "cell_type": "markdown", "id": "fe5d6f16-88bc-43cc-a503-ec7fe707ce45", "metadata": {}, "source": [ "Here, the lowest categorical axis indicates the primer, and the one above it indicates the siRNA. The y-axis represents the fold change over a scramble siRNA control. All four siRNAs show knockdown of their target genes but not the other transcripts examined. " ] }, { "cell_type": "markdown", "id": "342e330e-130a-4d6c-83e2-d781c3304856", "metadata": {}, "source": [ "## Further Reading\n", "\n", "1. Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45 (2001).\n", "\n", "2. Appropriate display of qPCR results and statistics - how to balance mathematical correctness and conventions? ResearchGate https://www.researchgate.net/post/Appropriate_display_of_qPCR_results_and_statistics-how_to_balance_mathematical_correctness_and_conventions.\n", "\n", "3. Can anyone help with calculating error in RT-qPCRs fold-change data? ResearchGate https://www.researchgate.net/post/Can_anyone_help_with_calculating_error_in_RT-qPCRs_fold-change_data.\n", "\n", "4. Wilhelm, J. & Pingoud, A. Real-Time Polymerase Chain Reaction. ChemBioChem 4, 1120–1128 (2003)." ] }, { "cell_type": "markdown", "id": "bad443a8-f230-4dfb-85e5-787efa4906ce", "metadata": {}, "source": [ "## Computing Environment" ] }, { "cell_type": "code", "execution_count": 9, "id": "e8dbe2e6-db45-4ad2-941f-5d5552dbe384", "metadata": { "tags": [] }, "outputs": [ { "name": "stdout", "output_type": "stream", "text": [ "Python implementation: CPython\n", "Python version : 3.9.17\n", "IPython version : 8.12.0\n", "\n", "equipt : 1.0.0\n", "jupyterlab: 3.6.3\n", "bokeh : 3.1.1\n", "holoviews : 1.16.2\n", "\n" ] } ], "source": [ "%load_ext watermark\n", "%watermark -v -p equipt,jupyterlab,bokeh,holoviews" ] } ], "metadata": { "kernelspec": { "display_name": "Python 3 (ipykernel)", "language": "python", "name": "python3" }, "language_info": { "codemirror_mode": { "name": "ipython", "version": 3 }, "file_extension": ".py", "mimetype": "text/x-python", "name": "python", "nbconvert_exporter": "python", "pygments_lexer": "ipython3", "version": "3.9.17" } }, "nbformat": 4, "nbformat_minor": 5 }