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1 """
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2 * Galaxy Version
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3
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4 * Copyright 2019 University of Liverpool
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5 * Author John Heap, Computational Biology Facility, UoL
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6 * Based on original scripts of Sara Silva Silva Pereira, Institute of Infection and Global Health, UoL
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7 *
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8 * Licensed under the Apache License, Version 2.0 (the "License");
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9 * you may not use this file except in compliance with the License.
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10 * You may obtain a copy of the License at
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11 *
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12 * http://www.apache.org/licenses/LICENSE-2.0
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13 *
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14 * Unless required by applicable law or agreed to in writing, software
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15 * distributed under the License is distributed on an "AS IS" BASIS,
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16 * WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
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17 * See the License for the specific language governing permissions and
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18 * limitations under the License.
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19 *
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20 """
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21
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22
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23 import subprocess
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24 import pandas as pd
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25 import re
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26 import os
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27 import sys
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28 import shutil
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29 # import matplotlib as mpl
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30 # mpl.use('Agg')
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31 import matplotlib.pyplot as plt
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32 import numpy as np
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33
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34
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35
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36
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37 # copies the user provided Fasta file to data/reference/file/file.fasta
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38 def uploadUserReferenceFastq(refFastq):
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39 refBase = os.path.basename(refFastq)
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40 ref = os.path.splitext(refBase)[0] # 'mydata/test.fasta' -> 'test'
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41 dir_path = os.path.dirname(os.path.realpath(__file__)) # directory of this file
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42 refDir = dir_path + "/data/Reference/" + ref #propose putting file in '/data/reference/ref/
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43 if not os.path.isdir(refDir): # if directory data/Reference/ref doesn't exist
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44 os.mkdir(refDir)
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45 refPath = refDir+"/"
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46 shutil.copy(refFastq, refPath + refBase) #copy reference file into the directory
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47 argString = "bowtie2-build " + refPath + refBase+" "+refPath+ref
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48 print("Building the bowtie2 reference files.")
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49 subprocess.call(argString, shell=True)
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50 return
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51
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52 def transcriptMapping(inputname, refFastq, forwardFN, reverseFN):
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53 # where is our Reference data?
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54 refBase = os.path.basename(refFastq)
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55 ref = os.path.splitext(refBase)[0]
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56 dir_path = os.path.dirname(os.path.realpath(__file__))
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57 refDir = dir_path + "/data/Reference/" + ref + "/"
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58 refName = refDir + ref
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59 # now have reference file so we can proceed with the transcript mapping via bowtie2
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60 argString = "bowtie2 -x "+refName+" -1 "+forwardFN+" -2 "+reverseFN+" -S "+inputname+".sam"
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61 print(argString)
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62 subprocess.call(argString, shell=True) #outputs a name.sam file
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63 return
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64
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65
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66
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67 def processSamFiles(inputname):
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68 cur_path = os.getcwd()
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69 samName = cur_path+"/"+inputname
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70 argString = "samtools view -bS "+inputname+".sam > "+samName+".bam"
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71 print(argString)
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72 subprocess.call(argString, shell=True)
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73
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74 argString = "samtools sort "+samName+".bam -o "+samName+".sorted"
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75 print("argstring = "+argString)
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76 subprocess.call(argString, shell=True)
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77
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78 argString = "samtools index "+samName+".sorted "+samName+".sorted.bai"
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79 print("argstring = " + argString)
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80 subprocess.call(argString, shell=True)
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81 return #we have saved out the relevent name.bam, name.sorted and name.sorted.bai files
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82
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83 # we will not have the .gtf file so call cufflinks without -G option
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84 def transcriptAbundance(inputname):
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85 argString = "cufflinks -o "+inputname+".cuff -u -p 8 "+inputname+".sorted"
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21
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86 subprocess.call(argString, shell=True)
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87 os.remove(inputname+".sorted") #remove name.sorted
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88 os.remove(inputname+".sorted.bai")
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89 os.remove(inputname+".bam")
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90 return
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91
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92 def transcriptsForBlast(name, refFastq):
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93 # quick and dirty just to see.
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94 refBase = os.path.basename(refFastq)
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95 ref = os.path.splitext(refBase)[0] # 'mydata/test.fasta' -> 'test'
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96 dir_path = os.path.dirname(os.path.realpath(__file__)) # directory of this file
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97 refPath = dir_path + "/data/Reference/" + ref + "/" + refBase # eg refPath = data/Reference/Trinity/Trinity.fasta
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98 # used for dirty # refPath = 'Trinity.fasta' # dirty one
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99 track_df = pd.read_csv(dir_path+'/' + name + '.cuff/genes.fpkm_tracking', sep='\t')
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100 names = track_df['locus']
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101 # print(len(names))
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102 # print(names[:5])
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103
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104 nlist = []
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105 for n in range(0,len(names)):
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106 i = names[n].find(':')
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107 nlist.append(names[n][:i])
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108 nameset = set(nlist) #get unique.
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109 with open(refPath, 'r') as myRef:
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110 refData = myRef.read()
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111 refData= refData+'\n>'
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112
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113 with open(name + '_for_blast.fa', 'w') as outfile:
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114 for trans_id in nameset:
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115 namepos = refData.find(trans_id)
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116 endpos = refData.find('>', namepos)
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117 outfile.write('>'+refData[namepos:endpos])
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118
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119 pass
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120
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121 def blastContigs(test_name,html_resource, database):
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122 db_path = database
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123 argString = "blastx -db "+db_path+" -query "+test_name+"_for_blast.fa -outfmt 10 -out "+test_name+"_blast.txt"
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124 print(argString)
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125 returncode = subprocess.call(argString, shell=True)
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126 if returncode != 0:
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127 return "Error in blastall"
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128 blast_df = pd.read_csv(""+test_name+"_blast.txt")
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129 blast_df.columns = ['qaccver', 'saccver', 'pident', 'length', 'mismatch', 'gapopen', 'qstart', 'qend', 'sstart', 'send', 'evalue','bitscore']
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130 blastResult_df = blast_df[(blast_df['pident']>=70) & (blast_df['length'] > 100) & (blast_df['evalue'] <=0.001) ]
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131 blastResult_df = blastResult_df[['qaccver', 'saccver', 'pident', 'evalue', 'bitscore']] #query accession.version, subject accession.version, Percentage of identical matches
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132 # need to allocate the transcripts (if allocated more than once to the phylotype with least error.
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133 transcripts = blastResult_df['qaccver']
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134 b_df = pd.DataFrame(columns=['qaccver', 'saccver', 'pident', 'evalue', 'bitscore'])
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135 transSet = set(transcripts)
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136 for t in transSet:
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137 temp_df = blastResult_df[(blastResult_df['qaccver'] == t)]
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138 # get one with smallest error value
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139 #print(t + ":")
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140 #print(temp_df)
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141 temp_df = temp_df.sort_values(by=['evalue'])
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142 b_df = b_df.append(temp_df.iloc[[0]])
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143
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144 b_df.sort_values(by=['qaccver'])
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145 b_df.to_csv(test_name + '_transcript.csv')
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146 return b_df
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147
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148
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149 def createMultiHTML(tdict,composite_df):
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150 labelList = composite_df.columns.tolist()
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151 htmlString = r"<html><title>T.vivax VAP (Transcriptomic Pathway(</title><body><div style='text-align:center'><h2><i>Trypanosoma vivax</i> Variant Antigen Profile</h2><h3>"
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152 htmlString += r"Sample name: "+tdict['name']
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153 htmlString += r"<br>Transcriptomic Analysis</h3></p>"
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154 htmlString += "<p style = 'margin-left:20%; margin-right:20%'>Legend: " \
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155 "Variant Antigen Profile of a <i>Trypanosoma vivax</i> transcriptomes. " \
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156 "Weighted Frequency reflects Phylotype abundance and is expressed as " \
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157 "phylotype frequencies adjusted for the combined transcript abundance. " \
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158 "Data was produced with VAPPER-Variant Antigen Profiler " \
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159 "(Silva Pereira et al., 2019).</p> "
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160 htmlString += r"<style> table, th, tr, td {border: 1px solid black; border-collapse: collapse;}</style>"
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161
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162 header = r"<table style='width:50%;margin-left:25%;text-align:center'><tr><th>Phylotype</th>"
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163 wLists = []
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164
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165 for j in range(1,len(labelList)):
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166 wLists.append(composite_df[labelList[j]])
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167 header += r"<th>" + str(labelList[j]) + "</th>"
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168
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169 htmlString += "</tr>\n" + header
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170 tabString = ""
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171 phyList = composite_df['Phylotype']
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172
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173
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174
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175 for i in range(0, len(composite_df)):
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176 tabString += "<tr><td>" + str(phyList[i]) + "</td>"
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177 for j in range(0,len(labelList)-1):
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178 #print(j)
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179 f = format(wLists[j][i], '.4f')
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180 tabString += "<td>" + str(f) + "</td>"
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181 tabString += "</tr>\n"
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182
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183 htmlString += tabString + "</table><br><br><br><br><br>"
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184 htmlString += r"<h3>Weighted Relative Frequencies of Detected Phylotypes.</h3>"
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185 imgString = r"<img src = '"+ tdict['name']+"_phylotypes.png' alt='Bar chart of phylotype variation' style='max-width:100%'><br><br>"
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186 htmlString += imgString
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187
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188 with open(tdict['html_file'], "w") as htmlfile:
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189 htmlfile.write(htmlString)
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190
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191
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192 def createHTML(tdict,sum_df):
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193 #assumes imgs are heatmap.png, dheatmap.png, vapPCA.png and already in htmlresource
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194 htmlString = r"<html><title>T.vivax VAP (Transcriptomic Pathway(</title><body><div style='text-align:center'><h2><i>Trypanosoma vivax</i> Variant Antigen Profile</h2><h3>"
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195 htmlString += r"Sample name: "+tdict['name']
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196 htmlString += r"<br>Transcriptomic Analysis</h3></p>"
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197 htmlString += "<p style = 'margin-left:20%; margin-right:20%'>Legend: " \
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198 "Variant Antigen Profile of a <i>Trypanosoma vivax</i> transcriptome. " \
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199 "Weighted Frequency reflects Phylotype abundance and is expressed as " \
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200 "phylotype frequencies adjusted for the combined transcript abundance. " \
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201 "Data was produced with VAPPER-Variant Antigen Profiler " \
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202 "(Silva Pereira et al., 2019).</p> "
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203 htmlString += r"<style> table, th, tr, td {border: 1px solid black; border-collapse: collapse;}</style>"
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204
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205 htmlString += r"<table style='width:50%;table-layout: auto; margin-left:25%;text-align:center'><tr><th>Phylotype</th>" \
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206 r"<th>Combined FPKM</th><th>Weighted Frequency</th></tr>"
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207 tabString = ""
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208 # flush out table with correct values
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209 phySeries = sum_df['Phylotype']
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210 # sacSeries = sum_df['saccver']
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211 fSeries = sum_df['FPKM']
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212 total = fSeries.sum()
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213 # print("Total="+str(total))
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214 for i in range(0, len(sum_df)):
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215 # print(phySeries[i])
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216 f = format(fSeries[i], '.2f')
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217 w = format(fSeries[i]/total, '.2f')
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218
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219 #w = format(weightList[i], '.4f')
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220
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221 tabString += "<tr><td>" + str(phySeries[i]) + "</td><td>" + str(f) + "</td><td>"+str(w)+"</tr>"
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222 htmlString += tabString + "</table><br><br><br><br><br>"
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223 htmlString += r"<h3>Weighted Relative Frequencies of Detected Phylotypes.</h3>"
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224 imgString = r"<img src = '"+ tdict['name']+"_phylotypes.png' alt='Bar chart of phylotype variation' style='max-width:100%'><br><br>"
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225 htmlString += imgString
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226
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227 with open(tdict['html_file'], "w") as htmlfile:
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228 htmlfile.write(htmlString)
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229
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230
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231
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232 def getPhyloNumber(sac):
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233 i = sac.find('_')
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234 return int(sac[1:i])
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235
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236 def combineFPMK(tdict):
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237 dir_path = os.path.dirname(os.path.realpath(__file__))+'/'
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238
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239 fpkm_df = pd.read_csv(dir_path++tdict['name']+'.cuff/genes.fpkm_tracking', sep='\t')
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240
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241 #fpkm_df = pd.read_csv('genes.fpkm_tracking',sep='\t')
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242 #print(fpkm_df.head())
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243 fpkm_df['locus'] = fpkm_df['locus'].apply(lambda names: names[:names.find(':')])
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244 #print(fpkm_df.head())
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245
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246 reducedBlast_df = pd.read_csv(dir_path + tdict['name']+'_transcript.csv')
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247 # reducedBlast_df = pd.read_csv('TrinityVT_transcript.csv')
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248 saccverSet = set(reducedBlast_df['saccver'])
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249 saccverList = list(saccverSet)
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250 saccverList.sort()
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251 # print(saccverList[:5])
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252 new_df = pd.DataFrame(columns=['qaccver','saccver','FPKM'])
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253 for sv in saccverList:
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254 #print(sv)
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255 temp_df = reducedBlast_df[reducedBlast_df['saccver'] == sv]
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256 qList = list(temp_df['qaccver'])
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257 for q in qList:
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258 f_df = fpkm_df[(fpkm_df['locus'] == q)]
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259 if len(f_df) > 1:
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260 print('WARNING MULTIPLE FPKM')
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261 new_fpkm=list(f_df['FPKM'])
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262 f = (new_fpkm[0])
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263 # print(f)
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264 new_df = new_df.append({'qaccver': q, 'saccver': sv, 'FPKM': f}, ignore_index=True)
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265 FPKMsum_df = new_df.groupby('saccver')['FPKM'].sum().reset_index()
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266
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267 FPKMsum_df['Phylotype'] = FPKMsum_df.apply(lambda row: getPhyloNumber(row['saccver']), axis=1)
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268 FPKMsum_df = FPKMsum_df.sort_values(by=['Phylotype'])
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269 FPKMsum_df = FPKMsum_df.reset_index(drop=True)
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270
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271 # print(FPKMsum_df)
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272 FPKMsum_df.to_csv('FPKM_sum.csv')
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273 FPKMsum2_df = FPKMsum_df.groupby('Phylotype')['FPKM'].sum().reset_index()
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274 FPKMsum2_df = FPKMsum2_df.sort_values(by=['Phylotype'])
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275
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276 # print(FPKMsum2_df)
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277 FPKMsum2_df.to_csv('FPKM_sum2.csv') # in case more than one entry for a particular phylotype
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278 return FPKMsum_df, FPKMsum2_df
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279
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280
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281
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282 def normalisef(f,max):
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283 return f/max
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284
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285 def getComposite_sum2(nameList,sum2_dfs):
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286 # lets get a composite sum2_df from all of the sum2_dfs
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287 phyList = []
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288
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289 for i in range(0, len(sum2_dfs)):
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290 total = sum2_dfs[i]['FPKM'].sum()
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291 sum2_dfs[i]['w'] = sum2_dfs[i].apply(lambda row: normalisef(row['FPKM'], total), axis=1)
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292 pSeries = sum2_dfs[i]['Phylotype']
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293 for p in pSeries:
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294 phyList.append(p) # get all the phylotypes in this one
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295 phyList = list(set(phyList))
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296 phyList.sort()
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297 composite_sum2_df = pd.DataFrame(phyList, columns=['Phylotype'])
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298 for i in range(0, len(sum2_dfs)):
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299 wList = []
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300 pindf = list(sum2_dfs[i]['Phylotype'])
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301 # print(pindf)
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302 for p in phyList:
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303 if p in pindf:
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304 df = sum2_dfs[i]
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305 w = df.loc[df['Phylotype'] == p, 'w'].iloc[0]
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306 else:
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307 w = 0
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308 wList.append(w)
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309 composite_sum2_df[nameList[i]] = wList
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310 #print(composite_sum2_df)
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311 #composite_sum2_df.to_csv('composite.csv')
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312 return composite_sum2_df
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313
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314
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315 def doMultiBarChart(tdict, composite_df): #array of multiple sum2_dfs
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316 labelList = composite_df.columns.tolist()
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317 sampnum = len(labelList)-1
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318 # need to arrange bars
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319 # number of phylotype = len(composite_df)
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320 #number of bars = (len(labelist)-1) +1 for space
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321 # ytick needs to ne
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322
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323 cmap = plt.cm.get_cmap('tab10')
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324 palette = [cmap(i) for i in range(cmap.N)]
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325 title = "Legend: Variant Antigen Profile of a $\itTrypanosoma$ $\itvivax$ transcriptomes. " \
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326 "Phylotype abundance is expressed as phylotype frequencies adjusted " \
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327 "for combined transcript abundance. " \
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328 "Data was produced with VAPPER-Variant Antigen Profiler (Silva Pereira et al., 2019)."
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329 width = 0.6
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330 ind = np.arange(width*sampnum/2, len(composite_df)*width*(sampnum+1), width*(sampnum+1))
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331 #print(ind)
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332 ysize = len(composite_df)*0.4
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333
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334 fig, ax = plt.subplots(figsize=(10,ysize))
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335
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336
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337 for s in range(1, len(labelList)):
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338 ax.barh(ind, composite_df[labelList[s]], width, color=palette[s], label=labelList[s])
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339 ind = ind + width
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340
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341 ax.set(yticks=np.arange(width*(sampnum+2)/2, len(composite_df)*width*(sampnum+1), width*(sampnum+1)), yticklabels=composite_df['Phylotype']) # , ylim=[(len(labelList)-1) * width - 1, len(composite_df)])
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342 ax.legend()
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343
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344
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345 ax.set_ylabel('Phylotype')
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346 ax.invert_yaxis() # labels read top-to-bottom
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347 ax.set_xlabel('Weighted Phylotype Frequency')
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348
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349 # plt.text(-0.3, -0.15, title, va="top", wrap="True")
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350 #plt.tight_layout()
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351
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352 plt.subplots_adjust(bottom=0.1, top=0.92, left=0.15, right=0.9)
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353 ax.set_title(title, x=0, wrap='True',ha='left',)
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354
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355 plt.savefig(tdict['html_resource'] + tdict['name']+"_phylotypes.png")
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356 if tdict['pdf'] == 'PDF_Yes':
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357 plt.savefig(tdict['html_resource'] + tdict['name']+"phylotypes.pdf")
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358 plt.show()
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359 pass
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360
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361
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362
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363 def doBarChart(tdict, sum2_df):
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364 cmap = plt.cm.get_cmap('tab20')
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365 palette = [cmap(i) for i in range(cmap.N)]
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366 title = "Legend: Variant Antigen Profile of a $\itTrypanosoma$ $\itvivax$ transcriptome. " \
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367 "Phylotype abundance is expressed as phylotype frequencies adjusted " \
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368 "for combined transcript abundance. " \
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369 "Data was produced with VAPPER-Variant Antigen Profiler (Silva Pereira et al., 2019)."
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370 # get a list of phylotype, create equivalent of saccver, get a list of
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371 maxFPKM = sum2_df['FPKM'].max()
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372 total = sum2_df['FPKM'].sum()
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373
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374 sum2_df['Normalised'] = sum2_df.apply(lambda row: normalisef(row['FPKM'], maxFPKM),axis=1)
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375 sum2_df['Weighted'] = sum2_df.apply(lambda row: normalisef(row['FPKM'], total),axis=1)
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376 pList = sum2_df['Phylotype']
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377 phList = []
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378 for p in pList:
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379 phList.append(str(p))
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380
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381 fList = sum2_df['Weighted']
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382 ysize = len(phList)*0.3
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383 fig, ax = plt.subplots(figsize=(10,ysize))
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384
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385 ax.barh(phList, fList, color=palette)
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386 ax.set_ylabel('Phylotype')
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387 ax.invert_yaxis() # labels read top-to-bottom
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388 ax.set_xlabel('Weighted Phylotype Frequency')
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389
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390 # plt.text(-0.3, -0.15, title, va="top", wrap="True")
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391 #plt.tight_layout()
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392 plt.subplots_adjust(bottom=0.1, top=0.9, left=0.15, right=0.9)
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393 ax.set_title(title, x=0, wrap='True',ha='left',)
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394
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395 plt.savefig(tdict['html_resource'] + tdict['name']+"_phylotypes.png")
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396 if tdict['pdf'] == 'PDF_Yes':
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397 plt.savefig(tdict['html_resource'] + tdict['name']+"phylotypes.pdf")
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398 # plt.show()
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399 pass
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400
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401 # argdict = {'name':2, 'pdfexport': 3, 'refFastq': 4, 'forward': 5, 'reverse': 6, 'html_file': 7, 'html_resource': 8}
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402
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403 def transcriptomicProcess(args,argdict):
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21
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404 dir_path = os.path.dirname(os.path.realpath(__file__))
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19
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405 tdict = {}
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406 tdict['name'] = args[argdict['name']]
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407 tdict['refFastq'] = args[argdict['refFastq']]
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408 tdict['forward'] = args[argdict['forward']]
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409 tdict['reverse'] = args[argdict['reverse']]
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21
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410 dir_path = os.path.dirname(os.path.realpath(__file__))
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411 tdict['vivax_trans_database'] = dir_path+'/data/vivax/Database/Phylotype_typeseqs.fas'
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19
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412 tdict['pdf'] = args[argdict['pdfexport']]
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413 tdict['html_file'] = args[argdict['html_file']]
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414 tdict['html_resource'] = args[argdict['html_resource']]
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415
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416 uploadUserReferenceFastq(tdict['refFastq'])
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417 transcriptMapping(tdict['name'], tdict['refFastq'], tdict['forward'], tdict['reverse']) #uses bowtie
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418 processSamFiles(tdict['name']) #uses samtools
|
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419 transcriptAbundance(tdict['name']) #uses cufflinks -> ?.cuff/*.*
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420 transcriptsForBlast(tdict['name'], tdict['refFastq']) #creates name+4blast.fa
|
21
|
421 blastContigs(tdict['name'], tdict['html_resource'], tdict['vivax_trans_database'])
|
19
|
422 sum_df, sum2_df = combineFPMK(tdict)
|
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423 doBarChart(tdict, sum2_df)
|
|
424 createHTML(tdict, sum_df)
|
|
425
|
|
426
|
|
427 if __name__ == "__main__":
|
|
428 exit()
|
|
429
|
|
430
|