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1 from mirbase_functions import *
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2 from mirbase_graphs import *
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3 import time
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4 from multiprocessing import Process, Queue, Lock, Pool, Manager, Value
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5 import subprocess
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6 import argparse
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7 import sys
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8
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9 subprocess.call(['mkdir','-p', 'split1','split2','split3','split4','Counts','Diff/temp_con','Diff/temp_tre','Diff/n_temp_con','Diff/n_temp_tre'])
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10
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11 parser = argparse.ArgumentParser()
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12 parser.add_argument("-analysis", "--anal", help="choose type of analysis", action="store")
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13 parser.add_argument("-con", "--control", help="input fastq file (controls)", nargs='+', default=[])
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14 parser.add_argument("-tre", "--treated", help="input fastq file (treated)", nargs='+', default=[] )
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15 parser.add_argument("-tool_dir", "--tool_directory", help="tool directory path", action="store")
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16 parser.add_argument("-gen", "--org_name", help="Organism", action="store")
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17 parser.add_argument("-f", "--flag", help="choose the database (MirBase,MirGene)", action="store")
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18 parser.add_argument("-percentage", "--per", help="Percentage of Samples", action="store")
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19 parser.add_argument("-counts", "--count", help="Counts for filtering", action="store")
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20 parser.add_argument("-name1", "--group1", help="Samples group 1", action="store")
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21 parser.add_argument("-name2", "--group2", help="Samples group 2", action="store")
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22 args = parser.parse_args()
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23
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24
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25 #################################################################################################################################################################################################################
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26
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27 if __name__ == '__main__':
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28
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29 starttime = time.time()
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30
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31 lock = Lock()
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32 manager = Manager()
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33
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34 # Download reference miRNA sequences from MirBase
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35 mature_mirnas=manager.list()
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36 ps_mature=Process(target=download_matures,args=(mature_mirnas,args.org_name))
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37 ps_mature.start()
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38
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39
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40 # Keep the names of the files and location paths
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41 args.control[0]=args.control[0][1:]
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42 args.control[len(args.control)-1][:-1]
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43 control = [(args.control[i:i+2]) for i in range(0, len(args.control), 2)]
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44
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45
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46 args.treated[0]=args.treated[0][1:]
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47 args.treated[len(args.treated)-1][:-1]
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48 treated = [(args.treated[i:i+2]) for i in range(0, len(args.treated), 2)]
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49
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50
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51 ############## Detection of templated isoforms ################
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52
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53
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54 # Initialization of the managers between the proccesses
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55 # First group of samples (controls)
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56 con_samples = manager.list() # Collapsed mirnas with the new names
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57 con_data= manager.list() # keeps all necessary data for the Database
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58 con_file_order=manager.list() # files' names ordered by processes
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59 con_names_seqs=manager.list() # keeps only mirna names and sequences
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60 deseq=manager.list()
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61 con_unmap_seq=manager.Value('i',0) # keeps unmap unique sequnces for the generation of a graph
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62 con_unmap_counts=manager.Value('i',0) # keeps unmap counts of sequnces for the generation of a graph
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63 con_mirna_names=manager.list() # keeps the names of mirnas
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64 ini_con_samples = manager.list() # filtered SAM files
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65
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66 # Second group of samples (treated)
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67 tre_samples = manager.list()
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68 tre_data = manager.list()
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69 tre_file_order = manager.list()
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70 tre_names_seqs=manager.list()
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71 deseq1=manager.list()
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72 tre_unmap_seq = manager.Value('i',0)
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73 tre_unmap_counts = manager.Value('i',0)
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74 tre_mirna_names=manager.list()
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75 ini_tre_samples = manager.list()
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76
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77 # Wait for the download of reference miRNA sequences
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78 ps_mature.join()
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79 mature_mirnas=list(mature_mirnas)
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80
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81 # Processing of the detected miRNAs from SAM files
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82 ps_sam = [Process(target=sam_edit,args=(mature_mirnas,path[1][:-1],path[0].split(",")[0],"c",lock,con_samples,con_data,con_file_order,con_unmap_seq,con_names_seqs,deseq,con_mirna_names,ini_con_samples,con_unmap_counts)) for path in control]
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83 ps_sam.extend([Process(target=sam_edit,args=(mature_mirnas,path[1][:-1],path[0].split(",")[0],"t",lock,tre_samples,tre_data,tre_file_order,tre_unmap_seq,tre_names_seqs,deseq1,tre_mirna_names,ini_tre_samples,tre_unmap_counts)) for path in treated])
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84
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85 # Wait for processing of SAM files to finish
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86 [p.start() for p in ps_sam]
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87 [p.join() for p in ps_sam]
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88
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89 # Generate a histogram
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90 ps_hist=[Process(target=hist_red,args=(ini_con_samples,'c',args.group1))]
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91 ps_hist.extend([Process(target=hist_red,args=(ini_tre_samples,'t',args.group2))])
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92 [x.start() for x in ps_hist]
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93
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94
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95 # Convert managers to lists
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96 con_samples = list(con_samples)
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97 tre_samples = list(tre_samples)
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98 con_file_order=list(con_file_order)
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99 tre_file_order=list(tre_file_order)
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100 deseq=list(deseq)
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101 deseq1=list(deseq1)
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102
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103 # Remove duplicates and sorting
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104 con_names_seqs=list(con_names_seqs)
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105 con_names_seqs.sort()
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106 con_names_seqs=list(con_names_seqs for con_names_seqs,_ in itertools.groupby(con_names_seqs))
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107
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108 tre_names_seqs=list(tre_names_seqs)
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109 tre_names_seqs.sort()
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110 tre_names_seqs=list(tre_names_seqs for tre_names_seqs,_ in itertools.groupby(tre_names_seqs))
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111
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112 # initialization of new managers
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113 new_con_file_order=manager.list()
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114 new_tre_file_order=manager.list()
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115 new_deseq=manager.list()
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116 new_deseq1=manager.list()
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117
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118 # add uncommon detected mirnas among the samples
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119 ps_un_mirnas=[Process(target=uncommon_mirnas,args=(sampp,con_names_seqs,lock,new_deseq,con_file_order[i],new_con_file_order)) for i,sampp in enumerate(deseq)]
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120 ps_un_mirnas.extend([Process(target=uncommon_mirnas,args=(sampp,tre_names_seqs,lock,new_deseq1,tre_file_order[i],new_tre_file_order)) for i,sampp in enumerate(deseq1)])
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121
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122 # Wait for processing of uncommon detected mirnas to finish
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123 [z.start() for z in ps_un_mirnas]
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124 [z.join() for z in ps_un_mirnas]
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125
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126 # Convert managers to lists
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127 new_deseq=list(new_deseq)
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128 new_deseq1=list(new_deseq1)
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129 con_file_order=list(new_con_file_order)
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130 tre_file_order=list(new_tre_file_order)
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131
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132 # Genereation of count matrices per group (controls - treated)
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133 control_group=[[x[0],x[2]] for x in new_deseq[0]]
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134 [control_group[i].append(y[i][1]) for i,_ in enumerate(control_group) for y in new_deseq]
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135
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136 treated_group=[[x[0],x[2]] for x in new_deseq1[0]]
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137 [treated_group[i].append(y[i][1]) for i,_ in enumerate(treated_group) for y in new_deseq1]
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138
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139 # Keep a copy of count matrices
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140 control_group_copy=copy.deepcopy(list(control_group))
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141 treated_group_copy=copy.deepcopy(list(treated_group))
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142
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143 # Initialization of managers
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144 merg_nam_control_group=manager.list()
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145 merg_nam_treated_group=manager.list()
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146
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147 # Merging of names different names for the same mirna sequence per group (controls, treated) to avoid duplicates
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148 ps_merge = [Process(target=merging_names,args=(control_group_copy,merg_nam_control_group))]
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149 ps_merge.extend([Process(target=merging_names,args=(treated_group_copy,merg_nam_treated_group))])
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150 [x.start() for x in ps_merge]
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151
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152
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153 # Add unique mirna sequences between groups (all groups will have the same amount of sequences)
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154 con_list=manager.list()
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155 tre_list=manager.list()
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156
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157 ps_bw = [Process(target=black_white,args=(con_names_seqs,tre_names_seqs,treated_group,tre_list))]
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158 ps_bw.extend([Process(target=black_white,args=(tre_names_seqs,con_names_seqs,control_group,con_list))])
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159 [x.start() for x in ps_bw]
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160 [x.join() for x in ps_bw]
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161
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162 control_group=list(con_list)
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163 treated_group=list(tre_list)
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164
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165 # Detection of duplications
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166 dupes=manager.list()
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167
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168 ps_dupes = Process(target=merging_dupes,args=(control_group,dupes))
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169 ps_dupes.start()
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170 ps_dupes.join()
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171
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172 dupes=list(dupes)
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173
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174 # Merging the duplications in one entry with all different names
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175 con_list=manager.list()
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176 tre_list=manager.list()
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177
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178 ps_ap_merg_dupes = [Process(target=apply_merging_dupes,args=(control_group,dupes,con_list))]
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179 ps_ap_merg_dupes.extend([Process(target=apply_merging_dupes,args=(treated_group,dupes,tre_list))])
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180 [x.start() for x in ps_ap_merg_dupes]
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181
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182 # Preparation of reference sequences (isodforms) for the detection of non template mirnas
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183 if args.anal=="2":
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184 all_iso = manager.list()
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185 ps_non_iso = Process(target=non_template_ref,args=(con_samples,tre_samples,all_iso))
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186 ps_non_iso.start()
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187
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188 # Finishing the process for merging
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189 [x.join() for x in ps_merge]
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190 merg_nam_control_group=list(merg_nam_control_group)
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191 merg_nam_treated_group=list(merg_nam_treated_group)
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192
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193 # Export the database and the graphs
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194 procs = [Process(target=DB_write,args=(x[0],x[1],x[2],x[3],1)) for x in con_data]
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195 procs.extend([Process(target=DB_write,args=(x[0],x[1],x[2],x[3],1)) for x in tre_data])
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196 procs.extend([Process(target=make_spider,args=(merg_nam_control_group,merg_nam_treated_group,args.group1,args.group2))])
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197
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198 if args.anal == "1":
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199 procs.extend([Process(target=pie_temp,args=(merg_nam_control_group,con_unmap_seq.value,con_unmap_counts.value,merg_nam_treated_group,tre_unmap_seq.value,tre_unmap_counts.value,group_name1,group_name2))])
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200
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201 [p.start() for p in procs]
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202
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203 # Export the pdf report file
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204 if args.anal=="1":
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205 [x.join() for x in ps_hist]
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206 [p.join() for p in procs]
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207 ps_pdf = Process(target=pdf_before_DE,args=(args.anal,args.group1,args.group2))
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208 ps_pdf.start()
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209
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210
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211 [x.join() for x in ps_ap_merg_dupes]
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212 control_group=list(con_list)
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213 treated_group=list(tre_list)
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214
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215 # Filters low count mirnas (otpional)
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216 if int(args.per)!=-1:
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217
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218 fil_con_group=manager.list()
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219 fil_tre_group=manager.list()
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220
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221 ps_low_counts = Process(target=filter_low_counts,args=(control_group,treated_group,fil_con_group,fil_tre_group,args.per,args.count))
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222 ps_low_counts.start()
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223 ps_low_counts.join()
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224
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225 fil_con_group=list(fil_con_group)
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226 fil_tre_group=list(fil_tre_group)
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227
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228 if "fil_con_group" not in locals() or "fil_con_group" not in globals():
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229 fil_con_group=control_group
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230 fil_tre_group=treated_group
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231
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232 # export count matrices
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233 ps_write = Process(target=write_main,args=(control_group, treated_group, fil_con_group, fil_tre_group, con_file_order,tre_file_order,1,args.group1,args.group2,args.per))
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234 ps_write.start()
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235
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236 # export counts files compatible with Deseq2 and EdgeR
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237 ps1_matrix = [Process(target=temp_counts_to_diff,args=(con_file_order,fil_con_group,"Diff/temp_con/"))]
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238 ps1_matrix.extend([Process(target=temp_counts_to_diff,args=(tre_file_order,fil_tre_group,"Diff/temp_tre/"))])
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239 [p.start() for p in ps1_matrix]
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240
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241 if args.anal=="1":
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242 ps_pdf.join()
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243 if args.anal=="2":
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244 [p.join() for p in procs]
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245 [x.join() for x in ps_hist]
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246
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247 ps_write.join()
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248 [p.join() for p in ps1_matrix]
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249
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250 ############################## Detection of non-template #######################################
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251
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252 starttime10 = time.time()
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253
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254 if args.anal == "2":
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255
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256 # Initialization of the managers between the proccesses
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257 # First group of samples (controls)
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258 n_con_data= manager.list()
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259 n_con_file_order=manager.list()
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260 n_con_names_seqs=manager.list()
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261 n_deseq=manager.list()
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262
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263 # Second group of samples (treated)
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264 n_tre_data = manager.list()
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265 n_tre_file_order = manager.list()
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266 n_tre_names_seqs=manager.list()
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267 n_deseq1=manager.list()
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268
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269 # Preparation of reference sequences
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270 new_ref_mirnas = list(mature_mirnas)
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271 ps_non_iso.join()
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272
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273 all_iso=list(all_iso)
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274 new_ref_mirnas.extend(all_iso)
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275
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276 # Processing of non template miRNAs from SAM files
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277 ps_sam = [Process(target=non_sam_edit,args=(new_ref_mirnas,path[1][:-1],path[0].split(",")[0],"c",lock,n_con_data,n_con_file_order,n_deseq,n_con_names_seqs)) for path in control]
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278 ps_sam.extend([Process(target=non_sam_edit,args=(new_ref_mirnas,path[1][:-1],path[0].split(",")[0],"t",lock,n_tre_data,n_tre_file_order,n_deseq1,n_tre_names_seqs)) for path in treated])
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279
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280 [p.start() for p in ps_sam]
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281 [p.join() for p in ps_sam]
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282
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283 # Convert managers to lists
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284 n_con_file_order=list(n_con_file_order)
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285 n_tre_file_order=list(n_tre_file_order)
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286 n_deseq=list(n_deseq)
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287 n_deseq1=list(n_deseq1)
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288
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289 # Remove duplicates and sorting
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290 n_con_names_seqs=list(n_con_names_seqs)
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291 n_con_names_seqs.sort()
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292 n_con_names_seqs=list(n_con_names_seqs for n_con_names_seqs,_ in itertools.groupby(n_con_names_seqs))
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293
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294 n_tre_names_seqs=list(n_tre_names_seqs)
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295 n_tre_names_seqs.sort()
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296 n_tre_names_seqs=list(n_tre_names_seqs for n_tre_names_seqs,_ in itertools.groupby(n_tre_names_seqs))
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297
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298 # initialization of new managers
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299 new_n_con_file_order=manager.list()
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300 new_n_tre_file_order=manager.list()
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301 n_new_deseq=manager.list()
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302 n_new_deseq1=manager.list()
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303
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304 # add uncommon detected mirnas among the samples
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305 ps_deseq=[Process(target=uncommon_mirnas,args=(sampp,n_con_names_seqs,lock,n_new_deseq,n_con_file_order[i],new_n_con_file_order)) for i,sampp in enumerate(n_deseq)]
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306 ps_deseq.extend([Process(target=uncommon_mirnas,args=(sampp,n_tre_names_seqs,lock,n_new_deseq1,n_tre_file_order[i],new_n_tre_file_order)) for i,sampp in enumerate(n_deseq1)])
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307
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308 # Wait for processing of uncommon detected mirnas to finish
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309 [x.start() for x in ps_deseq]
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310 [x.join() for x in ps_deseq]
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311
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312 # Convert managers to lists
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313 n_new_deseq=list(n_new_deseq)
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314 n_new_deseq1=list(n_new_deseq1)
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315 n_con_file_order=list(new_n_con_file_order)
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316 n_tre_file_order=list(new_n_tre_file_order)
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317
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318 # Genereation of count matrices per group (controls - treated)
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319 n_control_group=[[x[0],x[2]] for x in n_new_deseq[0]]
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320 [n_control_group[i].append(y[i][1]) for i,_ in enumerate(n_control_group) for y in n_new_deseq]
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321
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322 n_treated_group=[[x[0],x[2]] for x in n_new_deseq1[0]]
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323 [n_treated_group[i].append(y[i][1]) for i,_ in enumerate(n_treated_group) for y in n_new_deseq1]
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324
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325 # Keep a copy of count matrices
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326 n_control_group_copy=copy.deepcopy(list(n_control_group))
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327 n_treated_group_copy=copy.deepcopy(list(n_treated_group))
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328
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329 # Initialization of managers
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330 merg_nam_n_control_group=manager.list()
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331 merg_nam_n_treated_group=manager.list()
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332
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333 # Merging of names different names for the same mirna sequence per group (controls, treated) to avoid duplicates\
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334 ps_merge = [Process(target=merging_names,args=(n_control_group_copy,merg_nam_n_control_group))]
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335 ps_merge.extend([Process(target=merging_names,args=(n_treated_group_copy,merg_nam_n_treated_group))])
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336 [x.start() for x in ps_merge]
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337
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338 # Add unique mirna sequences between groups (all groups will have the same amount of sequences)
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339 n_con_list=manager.list()
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340 n_tre_list=manager.list()
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341
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342 ps_bw = [Process(target=black_white,args=(n_con_names_seqs,n_tre_names_seqs,n_treated_group,n_tre_list))]
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343 ps_bw.extend([Process(target=black_white,args=(n_tre_names_seqs,n_con_names_seqs,n_control_group,n_con_list))])
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344 [x.start() for x in ps_bw]
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345 [x.join() for x in ps_bw]
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346
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347 n_control_group=list(n_con_list)
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348 n_treated_group=list(n_tre_list)
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349
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350 # Detection of duplications
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351 n_dupes=manager.list()
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352
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353 ps_dupes = Process(target=merging_dupes,args=(n_control_group,n_dupes))
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354 ps_dupes.start()
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355 ps_dupes.join()
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356
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357 n_dupes=list(n_dupes)
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358
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359 # Merging the duplications in one entry with all different names
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360 n_con_list=manager.list()
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361 n_tre_list=manager.list()
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362
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363 ps_ap_merg_dupes = [Process(target=apply_merging_dupes,args=(n_control_group,n_dupes,n_con_list))]
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364 ps_ap_merg_dupes.extend([Process(target=apply_merging_dupes,args=(n_treated_group,n_dupes,n_tre_list))])
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365 [x.start() for x in ps_ap_merg_dupes]
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366
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367 # Finishing the process for merging
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368 [x.join() for x in ps_merge]
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369 merg_nam_n_control_group=list(merg_nam_n_control_group)
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370 merg_nam_n_treated_group=list(merg_nam_n_treated_group)
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371
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372 # Export the database and the graphs
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373 procs = [Process(target=DB_write,args=(x[0],x[1],x[2],x[3],2)) for x in n_con_data]
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374 procs.extend([Process(target=DB_write,args=(x[0],x[1],x[2],x[3],2)) for x in n_tre_data])
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375 procs.extend([Process(target=logo_seq_red,args=(merg_nam_n_control_group,'c',args.group1))])
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376 procs.extend([Process(target=logo_seq_red,args=(merg_nam_n_treated_group,'t',args.group2))])
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377 procs.extend([Process(target=pie_non_temp,args=(merg_nam_control_group,merg_nam_n_control_group,merg_nam_treated_group,merg_nam_n_treated_group,con_unmap_seq.value,tre_unmap_seq.value,con_unmap_counts.value,tre_unmap_counts.value,args.group1,args.group2))])
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378
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379 [p.start() for p in procs]
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380 [p.join() for p in procs]
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381
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382 procs1 = Process(target=pdf_before_DE,args=(args.anal,args.group1,args.group2))
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383 procs1.start()
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384
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385 [x.join() for x in ps_ap_merg_dupes]
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386 n_control_group=list(n_con_list)
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387 n_treated_group=list(n_tre_list)
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388
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389
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390 # Filters low count mirnas (otpional)
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391 if int(args.per)!=-1:
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392
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393 n_fil_con_group=manager.list()
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394 n_fil_tre_group=manager.list()
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395
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396 ps_low_counts = Process(target=filter_low_counts,args=(n_control_group,n_treated_group,n_fil_con_group,n_fil_tre_group,args.per,args.count))
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397 ps_low_counts.start()
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398 ps_low_counts.join()
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399
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400 n_fil_con_group=list(n_fil_con_group)
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401 n_fil_tre_group=list(n_fil_tre_group)
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402
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403 if "n_fil_con_group" not in locals() or "n_fil_con_group" not in globals():
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404 n_fil_con_group=n_control_group
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405 n_fil_tre_group=n_treated_group
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406
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407 ps_write = Process(target=write_main,args=(n_control_group, n_treated_group,n_fil_con_group, n_fil_tre_group, n_con_file_order, n_tre_file_order,2,args.group1,args.group2,args.per))
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408 ps_write.start()
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409
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410 ps1_matrix = [Process(target=nontemp_counts_to_diff,args=(n_con_file_order,n_fil_con_group,con_file_order,fil_con_group,"Diff/n_temp_con/"))]
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411 ps1_matrix.extend([Process(target=nontemp_counts_to_diff,args=(n_tre_file_order,n_fil_tre_group,tre_file_order,fil_tre_group,"Diff/n_temp_tre/"))])
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412 [p.start() for p in ps1_matrix]
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413
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414 ps_write.join()
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415 [p.join() for p in ps1_matrix]
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416 procs1.join()
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417 print('That took {} seconds'.format(time.time() - starttime10))
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418 print('That took {} seconds'.format(time.time() - starttime))
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419
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