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README.html
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<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml">
<head>
<meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
<meta http-equiv="Content-Style-Type" content="text/css" />
<meta name="generator" content="pandoc" />
<title>L. major UTR Analysis</title>
<style type="text/css">code{white-space: pre;}</style>
<meta name="viewport" content="width=device-width, initial-scale=1.0">
<!-- jQuery -->
<script src="https://cdnjs.cloudflare.com/ajax/libs/jquery/2.0.3/jquery.min.js"></script>
<script src="https://cdnjs.cloudflare.com/ajax/libs/jqueryui/1.10.3/jquery-ui.min.js"></script>
<!-- bootstrap -->
<!--<link href="https://netdna.bootstrapcdn.com/bootstrap/3.0.0/css/bootstrap.min.css" rel="stylesheet" id="style">-->
<script src="https://netdna.bootstrapcdn.com/bootstrap/3.0.0/js/bootstrap.min.js"></script>
<!-- highlight.js -->
<!--<link href="https://cdnjs.cloudflare.com/ajax/libs/highlight.js/7.3/styles/default.min.css" rel="stylesheet" id="code-style">-->
<script src="https://cdnjs.cloudflare.com/ajax/libs/highlight.js/7.3/highlight.min.js"></script>
<script>
hljs.LANGUAGES.r=function(a){var b="([a-zA-Z]|\\.[a-zA-Z.])[a-zA-Z0-9._]*";return{c:[a.HCM,{b:b,l:b,k:{keyword:"function if in break next repeat else for return switch while try tryCatch|10 stop warning require library attach detach source setMethod setGeneric setGroupGeneric setClass ...|10",literal:"NULL NA TRUE FALSE T F Inf NaN NA_integer_|10 NA_real_|10 NA_character_|10 NA_complex_|10"},r:0},{cN:"number",b:"0[xX][0-9a-fA-F]+[Li]?\\b",r:0},{cN:"number",b:"\\d+(?:[eE][+\\-]?\\d*)?L\\b",r:0},{cN:"number",b:"\\d+\\.(?!\\d)(?:i\\b)?",r:0},{cN:"number",b:"\\d+(?:\\.\\d*)?(?:[eE][+\\-]?\\d*)?i?\\b",r:0},{cN:"number",b:"\\.\\d+(?:[eE][+\\-]?\\d*)?i?\\b",r:0},{b:"`",e:"`",r:0},{cN:"string",b:'"',e:'"',c:[a.BE],r:0},{cN:"string",b:"'",e:"'",c:[a.BE],r:0}]}}(hljs); </script>
<!--<script type="text/javascript", src="https://yandex.st/highlightjs/7.3/languages/r.min.js"></script>-->
<!-- Manific Popup -->
<script src="https://cdnjs.cloudflare.com/ajax/libs/magnific-popup.js/0.8.9/jquery.magnific-popup.min.js"></script>
<script type="text/javascript"
src="https://cdn.mathjax.org/mathjax/latest/MathJax.js?config=TeX-AMS-MML_HTMLorMML">
</script>
<script defer="defer">
// Function to generate the dynamic table of contents
jQuery.fn.generate_TOC = function () {
var base = $(this[0]);
var selectors = ['h1', 'h2', 'h3', 'h4'];
var last_ptr = [{}, {}, {}, {}];
var anchors = {};
generate_anchor = function (text) {
var test = text.replace(/\W/g, '_');
while(test in anchors){
//if no suffix, add one
if(test.match(/_\d+$/) === null){
test = test + "_2";
}
//else generate unique id for duplicates by adding one to the suffix
else {
test = test.replace(/_(\d+)$/, function(match, number){ var num=+number+1; return("_" + num) });
}
}
anchors[test]=1;
return(test);
}
$(selectors.join(',')).filter(function(index) { return $(this).parent().attr("id") != 'header'; }).each(function () {
var heading = $(this);
var idx = selectors.indexOf(heading.prop('tagName').toLowerCase());
var itr = 0;
while (itr <= idx) {
if (jQuery.isEmptyObject(last_ptr[itr])) {
last_ptr[itr] = $('<ul>').addClass('nav');
if (itr === 0) {
base.append(last_ptr[itr])
} else {
if(last_ptr[itr-1].children('li').length === 0){
last_ptr[itr-1].append(last_ptr[itr]);
}
else {
last_ptr[itr - 1].children('li').last().append(last_ptr[itr]);
}
}
}
itr++;
}
var anchor = generate_anchor(heading.text());
heading.attr('id', anchor);
var a = $('<a>')
.text(heading.text())
.attr('href', '#' + anchor);
var li = $('<li>')
.append(a);
last_ptr[idx].append(li);
for (i = idx + 1; i < last_ptr.length; i++) {
last_ptr[i] = {};
}
});
}
/* run scripts when document is ready */
$(function() {
"use strict";
var $window = $(window);
var $body = $(document.body);
/* size of thumbnails */
var hidden_types = ['source']
var output_types = ['output', 'message', 'warning', 'error']
/* style tables */
$('table').addClass('table table-striped table-bordered table-hover table-condensed');
$('pre code').each(function(i, e) {
hljs.highlightBlock(e);
});
/* Magnific Popup */
$(".thumbnail").each(function(){
$(this).magnificPopup({
disableOn: 768,
closeOnContentClick: true,
type: 'image',
items: {
src: $(this).find('img').attr('src'),
}
});
});
function toggle_block(obj, show) {
var span = obj.find('span');
if(show === true){
span.removeClass('glyphicon-chevron-up').addClass('glyphicon-chevron-down');
obj.next('pre').slideDown();
}
else {
span.removeClass('glyphicon-chevron-down').addClass('glyphicon-chevron-up');
obj.next('pre').slideUp();
}
}
function toggle_thumbnails(imgs, show){
if(show === true){
imgs.parents().show()
imgs.slideDown();
}
else {
imgs.slideUp(400, function(){ $(this).parent().hide(); });
}
}
function global_toggle(obj){
var type = obj.attr('type');
var show = !obj.parent('li').hasClass('active');
if(show === true){
obj.parent('li').addClass('active');
}
else{
obj.parent('li').removeClass('active');
}
if(type == 'figure'){
toggle_thumbnails($('.thumbnail img'), show);
}
else {
$('.toggle.' + type).each(function() { toggle_block($(this), show); });
}
}
/* onclick toggle next code block */
$('.toggle').click(function() {
var span = $(this).find('span');
toggle_block($(this), !span.hasClass('glyphicon-chevron-down'));
return false
})
// global toggles
$('.toggle-global').click(function(){
var type = $(this).attr('type');
if(type === 'all-source'){
$('li a.source').each(function() {
global_toggle($(this));
});
}
else if(type === 'all-output'){
$.each(output_types, function(i, val){
console.log(val);
global_toggle($('li a.' + val));
});
}
else {
console.log($(this));
global_toggle($(this));
}
return false;
});
/* table of contents */
if($(['h1', 'h2', 'h3', 'h4'].join(',')).length > 0){
$('body > #wrap > .container > .row').append('<div class="col-md-2"><div id="toc" class="well sidebar sidenav affix hidden-print"/></div>');
$('#toc').generate_TOC();
}
$.each(hidden_types, function(i, type) {
$('li[type=' + type + ']').each(function(){ global_toggle($(this)); });
});
/* remove paragraphs with no content */
$('p:empty').remove();
$body.scrollspy({
target: '.sidebar',
});
/* theme switch */
$('.theme-switch').click(function(){
var css = $('link[title=' + $(this).attr('title') + ']');
$('#theme[rel=stylesheet]').attr('href', css.attr('href'));
$('.theme-switch').closest('li').removeClass('active');
$(this).closest('li').addClass('active');
return false;
});
/* code style switch */ //TODO use same function for both of these?
$('.highlight-switch').click(function(){
var css = $('link[title="' + $(this).attr('title') + '"]');
$('#highlight[rel=stylesheet]').attr('href', css.attr('href'));
$('.highlight-switch').closest('li').removeClass('active');
$(this).closest('li').addClass('active');
return false;
});
//TODO refresh on show/hide
$window.on('load', function () {
$body.scrollspy('refresh');
})
});
</script>
<style>
/* Knitr_bootstrap styles */
#header {
display: none !important;
visibility: hidden !important;
}
#wrap .container-fluid {
padding: 0;
overflow: hidden;
}
.toggle{
text-transform: capitalize;
}
.toggle-global{
text-transform: capitalize;
}
/* Sticky footer styles */
* {
margin:0;
}
html,
body {
height: 100%;
padding:0 !important;
/* The html and body elements cannot have any padding or margin. */
/*overflow-x: hidden;*/
}
/* Wrapper for page content to push down footer */
#wrap {
min-height: 100%;
height: auto !important;
height: 100%;
/* Negative indent footer by it's height */
margin: 0 auto -120px;
}
/* Set the fixed height of the footer here */
#push,
#footer {
height: 120px;
}
#footer {
text-align: center;
}
/* Top level subheader elements. These are the first nested items underneath a header element. */
.header li {
font-size: 20px;
}
/* Makes the font smaller for all subheader elements. */
.sub-header li {
font-size: 12px;
}
button.thumbnails {
margin-left:0px;
}
/*
* Side navigation
*
* Scrollspy and affixed enhanced navigation to highlight sections and secondary
* sections of docs content.
*/
/* By default it's not affixed in mobile views, so undo that */
.sidebar.affix {
position: static;
}
/* First level of nav */
.sidenav {
margin-top: 30px;
margin-bottom: 30px;
padding-top: 10px;
padding-bottom: 10px;
border-radius: 5px;
}
/* All levels of nav */
.sidebar .nav > li > a {
display: block;
padding: 5px 20px;
}
.sidebar .nav > li > a:hover,
.sidebar .nav > li > a:focus {
text-decoration: none;
border-right: 1px solid;
}
.sidebar .nav > .active > a,
.sidebar .nav > .active:hover > a,
.sidebar .nav > .active:focus > a {
font-weight: bold;
background-color: transparent;
border-right: 1px solid;
}
/* Nav: second level (shown on .active) */
.sidebar .nav .nav {
display: none; /* Hide by default, but at >768px, show it */
margin-bottom: 8px;
}
.sidebar .nav .nav > li > a {
padding-top: 3px;
padding-bottom: 3px;
padding-left: 30px;
font-size: 90%;
}
.sidebar .nav .nav .nav > li > a {
padding-left: 40px;
}
.sidebar .nav .nav .nav .nav > li > a {
padding-left: 50px;
}
/* Show and affix the side nav when space allows it */
@media screen and (min-width: 992px) {
.sidebar .nav > .active > ul {
display: block;
}
/* Widen the fixed sidebar */
.sidebar.affix,
.sidebar.affix-bottom {
width: 213px;
}
.sidebar.affix-top,
.sidebar.affix {
position: fixed; /* Undo the static from mobile first approach */
top: 30px;
}
.sidebar.affix-bottom {
position: absolute; /* Undo the static from mobile first approach */
}
.sidebar.affix-bottom .sidenav,
.sidebar.affix .sidenav {
margin-top: 0;
margin-bottom: 0;
}
}
@media screen and (min-width: 1200px) {
/* Widen the fixed sidebar again */
.sidebar.affix-bottom,
.sidebar.affix {
width: 263px;
}
}
#toc {
padding: 10px 0px;
margin:0;
border:0;
}
.panel pre {
margin: 0;
padding: 0;
border: 0;
}
button + pre {
margin: 0;
padding: 0;
}
pre code {
border-radius: 0;
}
/* Magnific Popup CSS */
.mfp-bg {
top: 0;
left: 0;
width: 100%;
height: 100%;
z-index: 1042;
overflow: hidden;
position: fixed;
background: #0b0b0b;
opacity: 0.8;
filter: alpha(opacity=80); }
.mfp-wrap {
top: 0;
left: 0;
width: 100%;
height: 100%;
z-index: 1043;
position: fixed;
outline: none !important;
-webkit-backface-visibility: hidden; }
.mfp-container {
text-align: center;
position: absolute;
width: 100%;
height: 100%;
left: 0;
top: 0;
padding: 0 8px;
-webkit-box-sizing: border-box;
-moz-box-sizing: border-box;
box-sizing: border-box; }
.mfp-container:before {
content: '';
display: inline-block;
height: 100%;
vertical-align: middle; }
.mfp-align-top .mfp-container:before {
display: none; }
.mfp-content {
position: relative;
display: inline-block;
vertical-align: middle;
margin: 0 auto;
text-align: left;
z-index: 1045; }
.mfp-inline-holder .mfp-content,
.mfp-ajax-holder .mfp-content {
width: 100%;
cursor: auto; }
.mfp-ajax-cur {
cursor: progress; }
.mfp-zoom-out-cur,
.mfp-zoom-out-cur .mfp-image-holder .mfp-close {
cursor: -moz-zoom-out;
cursor: -webkit-zoom-out;
cursor: zoom-out; }
.mfp-zoom {
cursor: pointer;
cursor: -webkit-zoom-in;
cursor: -moz-zoom-in;
cursor: zoom-in; }
.mfp-auto-cursor .mfp-content {
cursor: auto; }
.mfp-close,
.mfp-arrow,
.mfp-preloader,
.mfp-counter {
-webkit-user-select: none;
-moz-user-select: none;
user-select: none; }
.mfp-loading.mfp-figure {
display: none; }
.mfp-hide {
display: none !important; }
.mfp-preloader {
color: #cccccc;
position: absolute;
top: 50%;
width: auto;
text-align: center;
margin-top: -0.8em;
left: 8px;
right: 8px;
z-index: 1044; }
.mfp-preloader a {
color: #cccccc; }
.mfp-preloader a:hover {
color: white; }
.mfp-s-ready .mfp-preloader {
display: none; }
.mfp-s-error .mfp-content {
display: none; }
button.mfp-close,
button.mfp-arrow {
overflow: visible;
cursor: pointer;
background: transparent;
border: 0;
-webkit-appearance: none;
display: block;
padding: 0;
z-index: 1046;
-webkit-box-shadow: none;
box-shadow: none; }
button::-moz-focus-inner {
padding: 0;
border: 0; }
.mfp-close {
width: 44px;
height: 44px;
line-height: 44px;
position: absolute;
right: 0;
top: 0;
text-decoration: none;
text-align: center;
opacity: 0.65;
padding: 0 0 18px 10px;
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</head>
<body>
<div id="header">
<h1 class="title">L. major UTR Analysis</h1>
</div>
<div id="wrap">
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<div class="contents col-xs-12 col-md-10">
<h1>L. major UTR Length and Alternative Trans-splicing / Poly-adenylation Analysis</h1>
<h2>Overview</h2>
<p>The goal of this analysis is to parse the output from our <a href="https://github.com/khughitt/utr_analysis">UTR analysis pipeline</a>, perform some basic smoothing of the spliced leader and polya acceptor site counts and determine the most likely primary UTR boundaries for each gene where information is available. 5'- and 3'-UTR boundaries, along with some other useful information such as UTR GC- and CT-richness will be outputted in a format that is convenient for downstream analysis.</p>
<p>Finally, we will look for evidence of either alternative trans-splicing or poly-adenylation and attempt to visualize the prevelance and magnitude of these events across the different developmental stages.</p>
<h2>TODO</h2>
<ul>
<li>Include Trey's uORFs</li>
<li>Removal RNAs (SLRNA, etc)</li>
</ul>
<div class="row">
<a href='mailto:khughitt@umd.edu'>Keith Hughitt</a> (<time>2015-02-02</time>)
</div>
<h2>Settings</h2>
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<pre style=""><code class="source r"># Number of individual UTR plots to display for each developmental stage and
# UTR side. The purpose of these plots are to provide a sense of the data
# distribution and to visualize the effect of primary # site selection with and
# without smoothing.
max_plots = 10</code></pre>
</div>
<p><a href="README.rmd">view source</a></p>
<h2>Methods</h2>
<h3>Load data</h3>
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<pre style=""><code class="source r">library(GenomicRanges)
library(Biostrings)
library(rtracklayer)
library(ggplot2)
library(ggvis)
library(reshape2)
library(dplyr)
# Genome sequence and annotations from TriTrypDB (8.0)
gff = import.gff(file.path('/cbcb/lab/nelsayed/ref_data/lmajor_friedlin',
'/annotation/TriTrypDB-8.0_LmajorFriedlin.gff'),
version='3')
chromosomes = gff[gff$type == 'chromosome']
genes = gff[gff$type == 'gene']
# Load unannotated ORFs detected from ribosome profiling data
uorfs = import.gff(gzfile('input/lmajor_orfs_manual_2014-09-08_clean.gff.gz'), version='3')
uorfs$description = 'Unannotated ORF'
# Drop GFF columns not shared betwene TriTrypDB GFF and uORF GFF
keep_cols = intersect(colnames(mcols(genes)), colnames(mcols(uorfs)))
genes = genes[,keep_cols]
uorfs = uorfs[,keep_cols]
genes = append(genes, uorfs)
input_fasta = file.path(Sys.getenv("REF"),
"lmajor_friedlin/genome/TriTrypDB-8.0_LmajorFriedlin_Genome.fasta")
fasta = readDNAStringSet(input_fasta)
# Fix names (L. major chromosome identifiers)
names(fasta) = substring(names(fasta), 0, 7)
# Number of bases flanking CDS to scan for motifs when actual UTR length is not
# known; numbers are based on median 5' and 3' UTR lengths for L. major
default_5utr_width = 250
default_3utr_width = 575
# Taken from L. major UTR analysis output from 2014/11/14
procyclic_polya = import.gff(gzfile('input/lmajor_procyclic_ncrnas_removed_polya.gff.gz'), version='3')
metacyclic_polya = import.gff(gzfile('input/lmajor_metacyclic_ncrnas_removed_polya.gff.gz'), version='3')
amastigote_polya = import.gff(gzfile('input/lmajor_amastigote_ncrnas_removed_polya.gff.gz'), version='3')
procyclic_sl = import.gff(gzfile('input/lmajor_procyclic_ncrnas_removed_sl.gff.gz'), version='3')
metacyclic_sl = import.gff(gzfile('input/lmajor_metacyclic_ncrnas_removed_sl.gff.gz'), version='3')
amastigote_sl = import.gff(gzfile('input/lmajor_amastigote_ncrnas_removed_sl.gff.gz'), version='3')
# Filter noncoding RNAs
id_filter_string = 'rRNA|snRNA|snoRNA|SLRNA|TRNA|SRP'
noncoding_ids = genes$ID[grepl(id_filter_string, genes$ID)]
genes = genes[!genes$ID %in% noncoding_ids]
procyclic_polya = procyclic_polya[!procyclic_polya$Name %in% noncoding_ids,]
metacyclic_polya = metacyclic_polya[!metacyclic_polya$Name %in% noncoding_ids,]
amastigote_polya = amastigote_polya[!amastigote_polya$Name %in% noncoding_ids,]
procyclic_sl = procyclic_sl[!procyclic_sl$Name %in% noncoding_ids,]
metacyclic_sl = metacyclic_sl[!metacyclic_sl$Name %in% noncoding_ids,]
amastigote_sl = amastigote_sl[!amastigote_sl$Name %in% noncoding_ids,]
# Create output and build directories if needed
for (x in c('build', 'output')) {
if (!file.exists(x)) {
dir.create(x)
}
}</code></pre>
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<pre style=""><code class="source r">#
# find_peak
#
find_peak = function(gene, acceptor_sites, gene_strand, feature_type,
smoothed=FALSE, include_plot=FALSE, secondary=FALSE) {
# Determine orientation of feature relative to CDS
if ((feature_type == 'sl' && gene_strand == '+') ||
(feature_type == 'polya' && gene_strand == '-')) {
feature_side = 'left'
} else {
feature_side = 'right'
}
# Create a vector from the furthest SL site to the CDS boundary
if (feature_side == 'left') {
cds_boundary = start(gene)
raw_scores = rep(0, cds_boundary - min(start(acceptor_sites)))
rel_start = cds_boundary - start(acceptor_sites) + 1
} else {
cds_boundary = end(gene)
raw_scores = rep(0, max(start(acceptor_sites)) - cds_boundary)
rel_start = start(acceptor_sites) - cds_boundary + 1
}
# add scores at indices where acceptor sites were detected
raw_scores[rel_start] = score(acceptor_sites)
x = 1:length(raw_scores)
# create smooted version of scores (optional)
if (smoothed) {