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1 #!/usr/bin/env python2.7
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2 #
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3 # Copyright (C) 2014 INRA
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4 #
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5 # This program is free software: you can redistribute it and/or modify
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6 # it under the terms of the GNU General Public License as published by
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7 # the Free Software Foundation, either version 3 of the License, or
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8 # (at your option) any later version.
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9 #
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10 # This program is distributed in the hope that it will be useful,
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11 # but WITHOUT ANY WARRANTY; without even the implied warranty of
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12 # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
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13 # GNU General Public License for more details.
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14 #
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15 # You should have received a copy of the GNU General Public License
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16 # along with this program. If not, see <http://www.gnu.org/licenses/>.
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17 #
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18
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19 __author__ = 'Plateforme bioinformatique Toulouse and SIGENAE'
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20 __copyright__ = 'Copyright (C) 2015 INRA'
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21 __license__ = 'GNU General Public License'
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22 __version__ = '1.2.0'
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23 __email__ = 'frogs@inra.fr'
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24 __status__ = 'prod'
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25
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26 import os
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27 import sys
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28 import gzip
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29 import time
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30 import tarfile
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31 import argparse
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32
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33 CURRENT_DIR = os.path.dirname(os.path.abspath(__file__))
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34 # PATH
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35 BIN_DIR = os.path.abspath(os.path.join(os.path.dirname(CURRENT_DIR), "libexec"))
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36 os.environ['PATH'] = BIN_DIR + os.pathsep + os.environ['PATH']
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37 # PYTHONPATH
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38 LIB_DIR = os.path.abspath(os.path.join(os.path.dirname(CURRENT_DIR), "lib"))
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39 sys.path.append(LIB_DIR)
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40 if os.getenv('PYTHONPATH') is None: os.environ['PYTHONPATH'] = LIB_DIR
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41 else: os.environ['PYTHONPATH'] = os.environ['PYTHONPATH'] + os.pathsep + LIB_DIR
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42
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43 from frogsUtils import *
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44
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45
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46 ##################################################################################################################################################
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47 #
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48 # COMMAND LINES
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49 #
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50 ##################################################################################################################################################
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51 class Demultiplex(Cmd):
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52 """
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53 @summary : Demultiplex samples.
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54 """
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55 def __init__(self, R1_input_file, R2_input_file, barcode_file, mismatches, end, global_tmp_files, R1_output_files, R2_output_files, demultiplex_err_files1, demultiplex_err_files2, demultiplex_log):
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56 """
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57 @param R1_input_file : [str] Path to the R1 fastq file.
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58 @param R2_input_file : [str] Path to the R2 fastq file.
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59 @param barcode_file : [str] Path to barcodes and samples (one line by sample) description file. Line format : SAMPLE_NAME<TAB>BARCODE.
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60 @param mismatches : [int] Number of mismatches allowed
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61 @param end : [str] barcode ends ? forward : bol or reverse : eol (def bol)
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62 @param global_tmp_files : [str] Path for R1 and R2 files.
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63 @param R1_output_files : [list] Paths to the R1 fastq files (one by sample). User provides an empty list.
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64 @param R2_output_files : [list] Paths to the R2 fastq files (one by sample). User provides an empty list.
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65 @param demultiplex_err_files : [list] Paths to the files with ambiguous and unmatched reads. User provides an empty list.
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66 """
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67
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68 tmp_files = TmpFiles( global_tmp_files.tmp_dir )
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69
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70 tmp_folder = os.path.join( global_tmp_files.tmp_dir, global_tmp_files.prefix + "_tmp", tmp_files.prefix )
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71 global_tmp_files.dirs.append(tmp_folder)
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72 if not os.path.exists(tmp_folder):
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73 os.mkdir(tmp_folder)
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74 self.samples_names = list()
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75 # Update output data
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76 FH_barcode = open( barcode_file )
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77 for line in FH_barcode:
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78 sample_name, barcode = line.strip().rsplit(None, 1)
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79 # OI DEBUG
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80 print "Line is " + line
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81 print "Sample name is " + sample_name
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82 print "Barcode is " + barcode
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83 print "Append to R1_output_files file: " + tmp_folder + " " + sample_name + " " + '_R1.fastq'
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84 R1_output_files.append( os.path.join(tmp_folder, sample_name + '_R1.fastq') )
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85
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86 global_tmp_files.files.append(os.path.join(tmp_folder, sample_name + '_R1.fastq') )
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87 if R2_input_file != None:
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88 R2_output_files.append( os.path.join(tmp_folder, sample_name + '_R2.fastq') )
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89 global_tmp_files.files.append(os.path.join(tmp_folder, sample_name + '_R2.fastq'))
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90 self.samples_names.append( sample_name.replace(' ', '_') )
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91 FH_barcode.close()
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92 self.R1_input_file = R1_input_file
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93 self.ambiguous = os.path.join(tmp_folder, 'ambiguous_R1.fastq')
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94 self.unmatched = os.path.join(tmp_folder, 'unmatched_R1.fastq')
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95 demultiplex_err_files1.extend( [self.ambiguous,self.unmatched] )
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96 global_tmp_files.files.extend( [self.ambiguous,self.unmatched] )
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97 if R2_input_file != None:
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98 demultiplex_err_files2.extend( [os.path.join(tmp_folder, 'ambiguous_R2.fastq'),os.path.join(tmp_folder, 'unmatched_R2.fastq') ])
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99 global_tmp_files.files.extend( [os.path.join(tmp_folder, 'ambiguous_R2.fastq'),os.path.join(tmp_folder, 'unmatched_R2.fastq') ])
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100
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101 # Set class
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102 if R2_input_file != None:
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103 Cmd.__init__( self,
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104 'perl ' + os.environ['TOOL_DIRECTORY'] + '/splitbc.pl',
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105 'Demultiplex reads.',
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106 R1_input_file + ' ' + R2_input_file + ' --' + end + ' --bcfile ' + barcode_file + ' --mismatches ' + `mismatches` + ' --trim --no_adapt --prefix-r1 ' + os.path.join(tmp_folder, '%_R1.fastq') +\
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107 ' --prefix-r2 ' + os.path.join(tmp_folder, '%_R2.fastq') + ' >> ' + demultiplex_log,
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108 None )
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109 else:
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110 Cmd.__init__( self,
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111 'perl '+ os.environ['TOOL_DIRECTORY'] + '/splitbc.pl',
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112 'Demultiplex reads.',
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113 R1_input_file + ' --' + end + ' --bcfile ' + barcode_file + ' --mismatches ' + `mismatches` + ' --trim --no_adapt --prefix-r1 ' + os.path.join(tmp_folder, '%_R1.fastq') +\
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114 ' >> ' + demultiplex_log,
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115 None )
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116
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117 def parser(self, log_file):
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118 """
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119 @summary : Parse the command results to add information in log_file.
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120 @log_file : [str] Path to the sample process log file.
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121 """
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122 # Parse output
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123 nb_seq_before = get_fastq_nb_seq(self.R1_input_file)
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124 nb_seq_unmatched = get_fastq_nb_seq(self.unmatched)
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125 nb_seq_ambiguous = get_fastq_nb_seq(self.ambiguous)
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126 # Write result
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127 FH_log = Logger( log_file )
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128 FH_log.write( 'Results :\n' )
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129 FH_log.write( '\tnb seq before demultiplexing : ' + str(nb_seq_before) + '\n' )
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130 FH_log.write( '\tnb seq after process matched : ' + str(nb_seq_before - nb_seq_unmatched) + '\n' )
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131 FH_log.write( '\tnb seq after process non-ambiguous : ' + str(nb_seq_before - nb_seq_unmatched - nb_seq_ambiguous) + '\n' )
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132 FH_log.close()
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133
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134 def get_version(self):
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135 """
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136 @summary : Returns the program version number.
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137 @return : version number if this is possible, otherwise this method return 'unknown'.
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138 """
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139 return Cmd.get_version(self, 'stdout')
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140
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141
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142 class Archive(Cmd):
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143 """
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144 @summary : Creates an archive with files.
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145 """
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146 def __init__(self, archived_files, archive_path):
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147 """
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148 @param archived_files: [list] Files added in final archive.
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149 @param archive_path: [str] Path to the new archive.
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150 """
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151
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152 tmp_files=TmpFiles( os.path.dirname(archive_path) )
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153 tmp_folder = os.path.join( tmp_files.tmp_dir, tmp_files.prefix)
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154 tmp_files.dirs.append(tmp_folder)
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155 if not os.path.exists(tmp_folder):
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156 os.makedirs(tmp_folder)
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157
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158 if len(archived_files) == 0:
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159 raise Exception( "At least one file must be add to the archive '" + archive_path + "'." )
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160
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161 archived_basenames = list()
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162 for current in archived_files:
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163 if not os.path.dirname(current) == tmp_folder:
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164 os.rename(current, os.path.join(tmp_folder,os.path.basename(current)))
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165 tmp_files.files.append(os.path.join(tmp_folder,os.path.basename(current)))
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166 archived_basenames.append(os.path.basename(current))
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167 # OI DEBUG
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168 archive_path_present = "archive_path is file or directory: " + str(os.path.isfile(archive_path)) + " or " + str(os.path.isdir(archive_path))
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169 print archive_path_present
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170 tmp_folder_present = "tmp_folder is file or directory: " + str(os.path.isfile(tmp_folder)) + " or " + str(os.path.isdir(tmp_folder))
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171 print tmp_folder_present
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172 for files in archived_basenames:
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173 print "archived basename is " + files
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174 files_present = "archived basename is file or directory: " + str(os.path.isfile(files)) + " or " + str(os.path.isdir(files))
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175 print files_present
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176 # END OI DEBUG
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177
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178
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179
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180 Cmd.__init__( self,
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181 'tar',
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182 'Archives files.',
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183 '-zcf ' + archive_path + ' -C ' + tmp_folder + " " + " ".join(archived_basenames),
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184 None )
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185
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186 self.Files=tmp_files
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187
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188
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189 def parser(self,log_file):
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190 self.Files.deleteAll()
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191
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192
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193 ##################################################################################################################################################
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194 #
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195 # FUNCTIONS
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196 #
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197 ##################################################################################################################################################
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198 def is_gzip( file ):
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199 """
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200 @return: [bool] True if the file is gziped.
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201 @param file : [str] Path to processed file.
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202 """
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203 is_gzip = None
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204 FH_input = gzip.open( file )
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205 try:
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206 FH_input.readline()
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207 is_gzip = True
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208 except:
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209 is_gzip = False
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210 finally:
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211 FH_input.close()
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212 return is_gzip
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213
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214 def split_barcode_file( barcode_file, barcodes_file_list, global_tmp_files ):
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215 """
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216 @summary: In case of double multiplexe, split barcode file in one forward and multiple reverse barcode files
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217 @param barcode_file: [str] Path to the input barcode file
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218 @param barcodes_file_list: [list] List of path to the ouput barcode files
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219 @param out_dir: [str] path to the output directory to write barcode files
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220 """
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221 out_dir = global_tmp_files.tmp_dir
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222 barcode_input = open(barcode_file,"r")
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223 barcode_dict={}
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224 for l in barcode_input.readlines():
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225 [s,f,r]=l.strip().split()
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226 if not "forward_bc" in barcode_dict:
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227 barcode_dict["forward_bc"] = [f+"\t"+f]
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228 elif not f+"\t"+f in barcode_dict["forward_bc"]:
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229 barcode_dict["forward_bc"].append( f+"\t"+f)
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230 if not f+"_reverse_bc" in barcode_dict:
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231 barcode_dict[f+"_reverse_bc"] = [s+"\t"+r]
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232 else :
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233 barcode_dict[f+"_reverse_bc"].append(s+"\t"+r)
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234
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235 f=barcode_dict.pop("forward_bc")
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236 barcodes_file_list.append(os.path.join(out_dir,"forward_bc"))
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237 global_tmp_files.files.append(os.path.join(out_dir,"forward_bc"))
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238 FH_out = open(os.path.join(out_dir,"forward_bc"),"w")
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239 FH_out.write("\n".join(f)+"\n")
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240 FH_out.close()
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241
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242 for bc_file in barcode_dict:
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243 barcodes_file_list.append(os.path.join(out_dir,bc_file))
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244 global_tmp_files.files.append(os.path.join(out_dir,bc_file))
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245 FH_out = open(os.path.join(out_dir,bc_file),"w")
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246 FH_out.write("\n".join(barcode_dict[bc_file])+"\n")
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247 FH_out.close()
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248
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249 def get_fastq_nb_seq( fastq_file ):
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250 """
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251 @summary: Returns the number of sequences in fastq_file.
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252 @param fastq_file: [str] Path to the fastq file processed.
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253 @return: [int] The number of sequences.
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254 """
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255 FH_input = None
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256 if not is_gzip(fastq_file):
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257 FH_input = open( fastq_file )
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258 else:
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259 FH_input = gzip.open( fastq_file )
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260 nb_line = 0
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261 for line in FH_input:
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262 nb_line += 1
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263 FH_input.close()
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264 nb_seq = nb_line/4
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265 return nb_seq
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266
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267 def concat_files(list_input, output_file):
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268
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269 FH_out=open(output_file,"w")
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270 for f in list_input :
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271 FH_in = open(f)
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272 string=""
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273 i=0
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274 for line in FH_in:
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275 string+= line
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276 i+=1
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277 if i==2000 :
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278 FH_out.write(string)
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279 string=""
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280 i=0
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281 if i != 0:
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282 FH_out.write(string)
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283 FH_in.close()
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284 FH_out.close()
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285
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286 def summarise_results( summary_file, barcode_file, log_file ):
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287 """
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288 @summary: Writes one summary of results from several logs.
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289 @param summary_file: [str] The output file.
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290 @param log_files: [list] The list of path to log files (one log file by sample).
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291 """
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292 sample_dict=dict()
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293 FH_barcode= open(barcode_file)
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294 for line in FH_barcode:
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295 sample_dict[line.split()[0]]=0
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296
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297 FH_summary = open(summary_file, "w")
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298 FH_summary.write( "#sample\tcount\n")
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299 FH_log = open(log_file,"r")
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300 sample_dict["unmatched"]=0
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301 sample_dict["ambiguous"]=0
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302
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303 for line in FH_log.readlines():
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304 if line.startswith("Barcode") or line.startswith("total") :
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305 pass
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306 else :
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307 l=line.replace('(','\t').split()
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308 if l[0] in sample_dict:
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309 sample_dict[l[0]] += int(l[1])
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310
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311 for s in sample_dict:
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312 FH_summary.write(s + '\t' + str(sample_dict[s]) + '\n')
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313 FH_summary.close()
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314
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315
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316 ##################################################################################################################################################
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317 #
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318 # MAIN
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319 #
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320 ##################################################################################################################################################
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321 if __name__ == "__main__":
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322 # Manage parameters
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323 parser = argparse.ArgumentParser(
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324 description='Split by samples the reads in function of inner barcode.'
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325 )
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326 parser.add_argument('-m', '--mismatches', type=int, default=0, help="Number of mismatches allowed in barcode. [Default: %(default)s]")
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327 parser.add_argument('-e', '--end', type=str, default="bol", help="barcode is at the begining of the forward end (bol) or of the reverse (eol) or both (both). [Default: %(default)s]")
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328 parser.add_argument( '--debug', default=False, action='store_true', help="Keep temporary files to debug program." )
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329 parser.add_argument( '-v', '--version', action='version', version=__version__ )
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330 # Inputs
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331 group_input = parser.add_argument_group( 'Inputs' )
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332 group_input.add_argument( '--input-R1', required=True, help='The R1 sequence file with all samples (format: fastq).' )
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333 group_input.add_argument( '--input-R2', default=None, help='The R2 sequence file with all samples (format: fastq).' )
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334 group_input.add_argument( '--input-barcode', help='This file describes barcodes and samples (one line by sample). Line format : SAMPLE_NAME<TAB>BARCODE or SAMPLE_NAME<TAB>BARCODE_FW<TAB>BARCODE_RV.' )
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335 group_output = parser.add_argument_group( 'Outputs' )
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336 # Outputs
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337 group_output.add_argument( '--output-demultiplexed', default="demultiplexed_read.tar.gz", help='The tar file containing R1 files and R2 files for each sample (format: tar). [Default: %(default)s]' )
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338 group_output.add_argument( '--output-excluded', default="undemultiplexed_read.tar.gz", help='The tar file containing R1 files and R2 files not demultiplexed (format: tar). [Default: %(default)s]' )
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339 group_output.add_argument( '-s', '--summary', default='summary.tsv', help='TSV file with summary of filters results (format: TSV). [Default: %(default)s]')
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340 group_output.add_argument( '-l', '--log-file', default=sys.stdout, help='This output file will contain several information on executed commands.')
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341 args = parser.parse_args()
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342 prevent_shell_injections(args)
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343
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344 Logger.static_write(args.log_file, "## Application\nSoftware :" + sys.argv[0] + " (version : " + str(__version__) + ")\nCommand : " + " ".join(sys.argv) + "\n\n")
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345
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346 # Process
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347 R1_files = list()
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348 R2_files = list()
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349 tmp_barcode_files = list()
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350 tmp_R1_files = list()
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351 tmp_R2_files = list()
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352 demultiplex_err_files1 = list()
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353 demultiplex_err_files2 = list()
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354 excluded_R1_file = os.path.join(os.path.split(args.output_demultiplexed)[0],os.path.basename(args.input_R1)+"_excluded_demult")
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355 if args.input_R2 != None :
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356 excluded_R2_file = os.path.join(os.path.split(args.output_demultiplexed)[0],os.path.basename(args.input_R2)+"_excluded_demult")
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357 uniq_id = str(time.time()) + "_" + str(os.getpid())
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358
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359 tmp_files = TmpFiles( os.path.split(args.output_demultiplexed)[0] )
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360 demultiplex_log = tmp_files.add("Demult.log")
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361 tmp_folder=tmp_files.add_dir("tmp")
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362 os.mkdir(tmp_folder)
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363
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364 sample_list=[]
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365 try:
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366 # Process
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367 if args.end == "bol" or args.end == "eol" :
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368
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369 info="\n#Demultiplexing " + os.path.basename(args.input_R1)
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370 if args.input_R2 != None:
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371 info+= " and " + os.path.basename(args.input_R2)
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372 info += " with " + os.path.basename(args.input_barcode) + " in " + args.end + " strand\n"
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373 Logger.static_write(args.log_file,info)
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374 Demultiplex(args.input_R1, args.input_R2, args.input_barcode, args.mismatches, args.end, tmp_files, R1_files, R2_files, demultiplex_err_files1,demultiplex_err_files2, demultiplex_log).submit( args.log_file )
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375 # OI DEBUG
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376 for r1file in R1_files:
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377 Logger.static_write(args.log_file,"\n\t#Archive demultiplexed files is " + r1file + " \n")
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378
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379 else:
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380 split_barcode_file(args.input_barcode, tmp_barcode_files, tmp_files)
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381 info="\n#Demultiplexing " + os.path.basename(args.input_R1)
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382 if args.input_R2 != None:
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383 info+= " and " + os.path.basename(args.input_R2)
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384 info += " with " + os.path.basename(tmp_barcode_files[0]) + " in bol strand\n"
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385 Logger.static_write(args.log_file,info)
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386 Demultiplex(args.input_R1, args.input_R2, tmp_barcode_files[0], args.mismatches, "bol", tmp_files, tmp_R1_files, tmp_R2_files, demultiplex_err_files1,demultiplex_err_files2, demultiplex_log).submit( args.log_file )
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387 for idx,read1_file in enumerate(tmp_R1_files):
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388 bc = os.path.basename(read1_file).replace("_R1.fastq","")
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389 if os.path.join(tmp_files.tmp_dir,bc+"_reverse_bc") in tmp_barcode_files:
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390 if os.stat(tmp_R1_files[idx]).st_size != 0 :
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391 info="\n#Demultiplexing " + os.path.basename(tmp_R1_files[idx])
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392 if args.input_R2 != None:
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393 info+= " and " + os.path.basename(tmp_R2_files[idx])
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394 info += " with " + bc+"_reverse_bc" + " in eol strand\n"
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395 Logger.static_write(args.log_file,info)
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396 if args.input_R2 != None:
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397 Demultiplex(tmp_R1_files[idx], tmp_R2_files[idx], os.path.join(tmp_files.tmp_dir,bc+"_reverse_bc"), args.mismatches, "eol", tmp_files, R1_files, R2_files, demultiplex_err_files1, demultiplex_err_files2, demultiplex_log).submit( args.log_file )
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398 else:
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399 Demultiplex(tmp_R1_files[idx], None, os.path.join(tmp_files.tmp_dir,bc+"_reverse_bc"), args.mismatches, "eol", tmp_files, R1_files, R2_files, demultiplex_err_files1, demultiplex_err_files2, demultiplex_log).submit( args.log_file )
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400
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401 Logger.static_write(args.log_file,"\n#Summarising result\n")
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402 summarise_results( args.summary, args.input_barcode, demultiplex_log )
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403 Logger.static_write(args.log_file,"\n#Concatenation of undemultiplexed files 1\n")
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404 concat_files(demultiplex_err_files1, excluded_R1_file )
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405 if len(R2_files) > 0:
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406 Logger.static_write(args.log_file,"\n#Concatenation of undemultiplexed files 2\n")
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407 concat_files(demultiplex_err_files2, excluded_R2_file )
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408 Logger.static_write(args.log_file,"\n#Archive demultiplexed R1 and R2 files\n")
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409 Archive(R1_files + R2_files, args.output_demultiplexed).submit( args.log_file )
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410 Logger.static_write(args.log_file,"\n#Archive undemultiplexed R1 and R2 files\n")
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411 Archive([excluded_R1_file,excluded_R2_file], args.output_excluded).submit( args.log_file )
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412 else:
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413 Logger.static_write(args.log_file,"\n#Archive demultiplexed files\n")
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414 Archive(R1_files, args.output_demultiplexed).submit( args.log_file )
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415 Logger.static_write(args.log_file,"\n#Archive undemultiplexed files\n")
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416 Logger.static_write(args.log_file,"\n#Excluded_R1_file is " + excluded_R1_file + "\n")
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417 Archive([excluded_R1_file], args.output_excluded).submit( args.log_file )
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418
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419 # Remove temporary files
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420 finally:
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421 #OI DEBUG
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422 if not args.debug:
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423 pass
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424 #Logger.static_write(args.log_file,"\n#Removing temporary files\n")
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425 #tmp_files.deleteAll()
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