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0
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1 #!/usr/bin/env python
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2
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3 import argparse
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4 import os
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5 import shutil
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6 import subprocess
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7 import sys
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8 import shlex
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9 import tempfile
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10 import fileinput
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11 import fileinput
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12 from glob import glob
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13
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14 def stop_err( msg ):
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15 sys.stderr.write( "%s\n" % msg )
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16 sys.exit()
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17
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18 def __main__():
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19
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20 print 'tempfile_location',tempfile.gettempdir()
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21 #Parse Command Line
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22 parser = argparse.ArgumentParser(description='Wrapper for the bismark bisulfite mapper.')
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23 parser.add_argument( '-p', '--num-threads', dest='num_threads',
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24 type=int, default=4, help='Use this many threads to align reads. The default is 4.' )
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25
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26 parser.add_argument( '--bismark_path', dest='bismark_path', help='Path to the bismark perl scripts' )
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27
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28 parser.add_argument( '--bowtie2', action='store_true', default=False, help='Running bismark with bowtie2 and not with bowtie.' )
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29
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30 # input options
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31 parser.add_argument( '--own-file', dest='own_file', help='' )
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32 parser.add_argument( '-D', '--indexes-path', dest='index_path', help='Indexes directory; location of .ebwt and .fa files.' )
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33 parser.add_argument( '-O', '--output', dest='output' )
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34
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35
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36 parser.add_argument( '--output-report-file', dest='output_report_file' )
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37 parser.add_argument( '--suppress-header', dest='suppress_header', action="store_true" )
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38
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39 parser.add_argument( '--mate-paired', dest='mate_paired', action='store_true', help='Reads are mate-paired', default=False)
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40
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41
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42 parser.add_argument( '-1', '--mate1', dest='mate1',
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43 help='The forward reads file in Sanger FASTQ or FASTA format.' )
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44 parser.add_argument( '-2', '--mate2', dest='mate2',
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45 help='The reverse reads file in Sanger FASTQ or FASTA format.' )
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46
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47 parser.add_argument( '--output-unmapped-reads', dest='output_unmapped_reads',
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48 help='Additional output file with unmapped reads (single-end).' )
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49 parser.add_argument( '--output-unmapped-reads-l', dest='output_unmapped_reads_l',
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50 help='File name for unmapped reads (left, paired-end).' )
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51 parser.add_argument( '--output-unmapped-reads-r', dest='output_unmapped_reads_r',
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52 help='File name for unmapped reads (right, paired-end).' )
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53
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54
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55 parser.add_argument( '--output-suppressed-reads', dest='output_suppressed_reads',
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56 help='Additional output file with suppressed reads (single-end).' )
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57 parser.add_argument( '--output-suppressed-reads-l', dest='output_suppressed_reads_l',
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58 help='File name for suppressed reads (left, paired-end).' )
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59 parser.add_argument( '--output-suppressed-reads-r', dest='output_suppressed_reads_r',
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60 help='File name for suppressed reads (right, paired-end).' )
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61 parser.add_argument( '--stdout', dest='output_stdout',
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62 help='File name for the standard output of bismark.' )
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63
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64
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65 parser.add_argument( '--single-paired', dest='single_paired',
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66 help='The single-end reads file in Sanger FASTQ or FASTA format.' )
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67
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68 parser.add_argument( '--fastq', action='store_true', help='Query filetype is in FASTQ format')
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69 parser.add_argument( '--fasta', action='store_true', help='Query filetype is in FASTA format')
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70 parser.add_argument( '--phred64-quals', dest='phred64', action="store_true" )
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71
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72
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73 parser.add_argument( '--skip-reads', dest='skip_reads', type=int )
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74 parser.add_argument( '--qupto', type=int)
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75
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76
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77 # paired end options
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78 parser.add_argument( '-I', '--minins', dest='min_insert' )
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79 parser.add_argument( '-X', '--maxins', dest='max_insert' )
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80 parser.add_argument( '--no-mixed', dest='no_mixed', action="store_true" )
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81 parser.add_argument( '--no-discordant', dest='no_discordant', action="store_true" )
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82
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83 #parse general options
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84 # default 20
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85 parser.add_argument( '--seed-len', dest='seed_len', type=int)
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86 # default 15
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87 parser.add_argument( '--seed-extention-attempts', dest='seed_extention_attempts', type=int )
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88 # default 0
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89 parser.add_argument( '--seed-mismatches', dest='seed_mismatches', type=int )
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90 # default 2
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91 parser.add_argument( '--max-reseed', dest='max_reseed', type=int )
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92 """
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93 # default 70
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94 parser.add_argument( '--maqerr', dest='maqerr', type=int )
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95 """
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96
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97 """
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98 The number of megabytes of memory a given thread is given to store path
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99 descriptors in --best mode. Best-first search must keep track of many paths
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100 at once to ensure it is always extending the path with the lowest cumulative
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101 cost. Bowtie tries to minimize the memory impact of the descriptors, but
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102 they can still grow very large in some cases. If you receive an error message
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103 saying that chunk memory has been exhausted in --best mode, try adjusting
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104 this parameter up to dedicate more memory to the descriptors. Default: 512.
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105 """
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106 parser.add_argument( '--chunkmbs', type=int, default=512 )
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107
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108 args = parser.parse_args()
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109
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110 # Create bismark index if necessary.
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111 index_dir = ""
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112 if args.own_file:
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113 """
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114 Create a temporary index with the offered files from the user.
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115 Utilizing the script: bismark_genome_preparation
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116 bismark_genome_preparation --bowtie2 hg19/
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117 """
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118 tmp_index_dir = tempfile.mkdtemp()
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119 index_path = os.path.join( tmp_index_dir, '.'.join( os.path.split( args.own_file )[1].split( '.' )[:-1] ) )
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120 try:
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121 """
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122 Create a hard link pointing to args.own_file named 'index_path'.fa.
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123 """
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124 os.symlink( args.own_file, index_path + '.fa' )
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125 except Exception, e:
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126 if os.path.exists( tmp_index_dir ):
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127 shutil.rmtree( tmp_index_dir )
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128 stop_err( 'Error in linking the reference database.\n' + str( e ) )
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129 # bismark_genome_preparation needs the complete path to the folder in which the database is stored
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130 if args.bowtie2:
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131 cmd_index = 'bismark_genome_preparation --bowtie2 %s ' % ( tmp_index_dir )
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132 else:
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133 cmd_index = 'bismark_genome_preparation %s ' % ( tmp_index_dir )
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134 if args.bismark_path:
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135 if os.path.exists(args.bismark_path):
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136 # add the path to the bismark perl scripts, that is needed for galaxy
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137 cmd_index = os.path.join(args.bismark_path, cmd_index)
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138 else:
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139 # assume the same directory as that script
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140 cmd_index = 'perl %s' % os.path.join(os.path.realpath(os.path.dirname(__file__)), cmd_index)
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141 try:
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142 tmp = tempfile.NamedTemporaryFile( dir=tmp_index_dir ).name
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143 tmp_stderr = open( tmp, 'wb' )
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144 proc = subprocess.Popen( args=cmd_index, shell=True, cwd=tmp_index_dir, stdout=open(os.devnull, 'wb'), stderr=tmp_stderr.fileno() )
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145 returncode = proc.wait()
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146 tmp_stderr.close()
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147 # get stderr, allowing for case where it's very large
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148 tmp_stderr = open( tmp, 'rb' )
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149 stderr = ''
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150 buffsize = 1048576
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151 try:
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152 while True:
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153 stderr += tmp_stderr.read( buffsize )
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154 if not stderr or len( stderr ) % buffsize != 0:
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155 break
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156 except OverflowError:
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157 pass
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158 tmp_stderr.close()
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159 if returncode != 0:
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160 raise Exception, stderr
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161 except Exception, e:
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162 if os.path.exists( tmp_index_dir ):
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163 shutil.rmtree( tmp_index_dir )
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164 stop_err( 'Error indexing reference sequence\n' + str( e ) )
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165 index_dir = tmp_index_dir
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166 else:
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167 # bowtie path is the path to the index directory and the first path of the index file name
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168 index_dir = os.path.dirname( args.index_path )
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169
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170 # Build bismark command
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171
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172 """
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173 Bismark requires a large amount of temporary disc space. If that is not available, for example on a cluster you can hardcode the
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174 TMP to some larger space. It's not recommended but it works.
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175 """
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176 #tmp_bismark_dir = tempfile.mkdtemp( dir='/data/0/galaxy_db/tmp/' )
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177 tmp_bismark_dir = tempfile.mkdtemp()
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178 output_dir = os.path.join( tmp_bismark_dir, 'results')
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179 cmd = 'bismark %(args)s --bam --temp_dir %(tmp_bismark_dir)s -o %(output_dir)s --quiet %(genome_folder)s %(reads)s'
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180
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181 if args.fasta:
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182 # he query input files (specified as mate1,mate2 or singles) are FastA
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183 cmd = '%s %s' % (cmd, '--fasta')
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184 elif args.fastq:
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185 cmd = '%s %s' % (cmd, '--fastq')
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186
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187 if args.bismark_path:
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188 # add the path to the bismark perl scripts, that is needed for galaxy
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189 if os.path.exists(args.bismark_path):
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190 cmd = os.path.join(args.bismark_path, cmd)
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191 else:
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192 # assume the same directory as that script
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193 cmd = 'perl %s' % os.path.join(os.path.realpath(os.path.dirname(__file__)), cmd)
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194
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195 arguments = {
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196 'genome_folder': index_dir,
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197 'args': '',
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198 'tmp_bismark_dir': tmp_bismark_dir,
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199 'output_dir': output_dir,
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200 }
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201
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202 additional_opts = ''
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203 # Set up the reads
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204 if args.mate_paired:
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205 # paired-end reads library
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206 reads = '-1 %s ' % ( args.mate1 )
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207 reads += ' -2 %s ' % ( args.mate2 )
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208 additional_opts += ' -I %s -X %s ' % (args.min_insert, args.max_insert)
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209 else:
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210 # single paired reads library
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211 reads = ' %s ' % ( args.single_paired )
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212
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213
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214 if not args.bowtie2:
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215 # use bowtie specific options
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216 #additional_opts += ' --best ' # bug in bismark, --best is not available as option. Only --non-best, best-mode is activated by default
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217 if args.seed_mismatches:
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218 # --seedmms
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219 additional_opts += ' -n %s ' % args.seed_mismatches
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220 if args.seed_len:
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221 # --seedlen
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222 additional_opts += ' -l %s ' % args.seed_len
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223
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224 # alignment options
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225 if args.bowtie2:
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226 additional_opts += ' -p %s --bowtie2 ' % args.num_threads
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227 if args.seed_mismatches:
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228 additional_opts += ' -N %s ' % args.seed_mismatches
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229 if args.seed_len:
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230 additional_opts += ' -L %s ' % args.seed_len
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231 if args.seed_extention_attempts:
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232 additional_opts += ' -D %s ' % args.seed_extention_attempts
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233 if args.max_reseed:
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234 additional_opts += ' -R %s ' % args.max_reseed
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235 if args.no_discordant:
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236 additional_opts += ' --no-discordant '
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237 if args.no_mixed:
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238 additional_opts += ' --no-mixed '
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239 """
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240 if args.maqerr:
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241 additional_opts += ' --maqerr %s ' % args.maqerr
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242 """
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243 if args.skip_reads:
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244 additional_opts += ' --skip %s ' % args.skip_reads
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245 if args.qupto:
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246 additional_opts += ' --qupto %s ' % args.qupto
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247 if args.phred64:
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248 additional_opts += ' --phred64-quals '
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249 if args.suppress_header:
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250 additional_opts += ' --sam-no-hd '
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251 if args.output_unmapped_reads or ( args.output_unmapped_reads_l and args.output_unmapped_reads_r):
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252 additional_opts += ' --un '
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253 if args.output_suppressed_reads or ( args.output_suppressed_reads_l and args.output_suppressed_reads_r):
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254 additional_opts += ' --ambiguous '
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255
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256 arguments.update( {'args': additional_opts, 'reads': reads} )
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257
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258 # Final bismark command:
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259 cmd = cmd % arguments
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260 print 'bismark_cmd:', cmd
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261 #sys.stderr.write( cmd )
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262 #sys.exit(1)
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263 # Run
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264 try:
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265 tmp_out = tempfile.NamedTemporaryFile().name
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266 tmp_stdout = open( tmp_out, 'wb' )
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267 tmp_err = tempfile.NamedTemporaryFile().name
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268 tmp_stderr = open( tmp_err, 'wb' )
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269 proc = subprocess.Popen( args=cmd, shell=True, cwd=".", stdout=tmp_stdout, stderr=tmp_stderr )
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270 returncode = proc.wait()
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271
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272 if returncode != 0:
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273 tmp_stdout.close()
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274 tmp_stderr.close()
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275 # get stderr, allowing for case where it's very large
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276 tmp_stderr = open( tmp_err, 'rb' )
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277 stderr = ''
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278 buffsize = 1048576
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279 try:
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280 while True:
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281 stderr += tmp_stderr.read( buffsize )
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282 if not stderr or len( stderr ) % buffsize != 0:
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283 break
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284 except OverflowError:
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285 pass
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286
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287 raise Exception, stderr
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288
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289 tmp_stdout.close()
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290 tmp_stderr.close()
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291
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292 # TODO: look for errors in program output.
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293 except Exception, e:
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294 stop_err( 'Error in bismark:\n' + str( e ) )
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295
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296 # collect and copy output files
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297 if args.output_report_file:
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298 output_report_file = open(args.output_report_file, 'w+')
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299 for line in fileinput.input(glob( os.path.join( output_dir, '*report.txt') )):
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300 output_report_file.write(line)
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301 output_report_file.close()
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302
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303
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304 if args.output_suppressed_reads:
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305 shutil.move( glob(os.path.join( output_dir, '*ambiguous_reads.txt'))[0], args.output_suppressed_reads )
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306 if args.output_suppressed_reads_l:
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307 shutil.move( glob(os.path.join( output_dir, '*ambiguous_reads_1.txt'))[0], args.output_suppressed_reads_l )
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308 if args.output_suppressed_reads_r:
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309 shutil.move( glob(os.path.join( output_dir, '*ambiguous_reads_2.txt'))[0], args.output_suppressed_reads_r )
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310
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311 if args.output_unmapped_reads:
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312 shutil.move( glob(os.path.join( output_dir, '*unmapped_reads.txt'))[0], args.output_unmapped_reads )
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313 if args.output_unmapped_reads_l:
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314 shutil.move( glob(os.path.join( output_dir, '*unmapped_reads_1.txt'))[0], args.output_unmapped_reads_l )
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315 if args.output_unmapped_reads_r:
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316 shutil.move( glob(os.path.join( output_dir, '*unmapped_reads_2.txt'))[0], args.output_unmapped_reads_r )
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317
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318 try:
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319 """
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320 merge all bam files
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321 """
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322 #tmp_out = tempfile.NamedTemporaryFile( dir=output_dir ).name
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323 tmp_stdout = open( tmp_out, 'wab' )
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324 tmp_err = tempfile.NamedTemporaryFile( dir=output_dir ).name
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325 tmp_stderr = open( tmp_err, 'wb' )
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326
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327 tmp_res = tempfile.NamedTemporaryFile( dir= output_dir).name
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328
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329 bam_files = glob( os.path.join( output_dir, '*.bam') )
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330 if len( bam_files ) > 1:
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331 cmd = 'samtools merge -@ %s -f - %s ' % ( args.num_threads, ' '.join( bam_files ) )
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332
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333 p1 = subprocess.Popen( args=shlex.split( cmd ), stdout=subprocess.PIPE )
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334 proc = subprocess.Popen( ['samtools', 'sort', '-@', str(args.num_threads), '-', tmp_res], stdin=p1.stdout, stdout=tmp_stdout, stderr=tmp_stderr )
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335 else:
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336 proc = subprocess.Popen( ['samtools', 'sort', '-@', str(args.num_threads), bam_files[0], tmp_res], stdout=tmp_stdout, stderr=tmp_stderr )
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337
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338 returncode = proc.wait()
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339 tmp_stdout.close()
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340 tmp_stderr.close()
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341 if returncode != 0:
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342 raise Exception, open( tmp_stderr.name ).read()
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343
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344 bam_path = "%s.bam" % tmp_res
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345 if os.path.exists( bam_path ):
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346 shutil.copy( bam_path, args.output )
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347 else:
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348 stop_err( 'BAM file no found:\n' + str( bam_path ) )
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349
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350 # TODO: look for errors in program output.
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351 except Exception, e:
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352 stop_err( 'Error in merging bam files:\n' + str( e ) )
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353
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354
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355 if args.output_stdout:
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356 # copy the temporary saved stdout from bismark
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357 shutil.move( tmp_out, args.output_stdout )
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358
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359 # Clean up temp dirs
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360 if args.own_file:
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361 if os.path.exists( tmp_index_dir ):
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362 shutil.rmtree( tmp_index_dir )
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363 if os.path.exists( tmp_bismark_dir ):
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364 shutil.rmtree( tmp_bismark_dir )
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365 if os.path.exists( output_dir ):
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366 shutil.rmtree( output_dir )
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367
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368 if __name__=="__main__": __main__()
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