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1 #!/usr/bin/env python
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2 # small RNA oriented bowtie wrapper in cascade for small RNA data set genome annotation
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3 # version 0.9 13-6-2014
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4 # Usage sRbowtie_cascade.py see Parser() for valid arguments
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5 # Christophe Antoniewski <drosofff@gmail.com>
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6
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7 import sys, os, subprocess, tempfile, shutil, argparse
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8 from collections import defaultdict
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9
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10 def Parser():
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11 the_parser = argparse.ArgumentParser()
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12 the_parser.add_argument('--output', action="store", type=str, help="output file")
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13 the_parser.add_argument('--num-threads', dest="num_threads", action="store", type=str, help="number of bowtie threads")
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14 the_parser.add_argument('--mismatch', action="store", type=str, help="number of mismatches allowed")
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15 the_parser.add_argument('--indexing-flags', dest="indexing_flags", nargs='+', help="whether the index should be generated or not by bowtie-buid")
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16 the_parser.add_argument('--index',nargs='+', help="paths to indexed or fasta references")
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17 the_parser.add_argument('--indexName',nargs='+', help="Names of the indexes")
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18 the_parser.add_argument('--input',nargs='+', help="paths to multiple input files")
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19 the_parser.add_argument('--label',nargs='+', help="labels of multiple input files")
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20 args = the_parser.parse_args()
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21 return args
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22
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23 def stop_err( msg ):
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24 sys.stderr.write( '%s\n' % msg )
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25 sys.exit()
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26
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27 def bowtie_squash(fasta):
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28 tmp_index_dir = tempfile.mkdtemp() # make temp directory for bowtie indexes
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29 ref_file = tempfile.NamedTemporaryFile( dir=tmp_index_dir )
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30 ref_file_name = ref_file.name
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31 ref_file.close() # by default, delete the temporary file, but ref_file.name is now stored in ref_file_name
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32 os.symlink( fasta, ref_file_name ) # symlink between the fasta source file and the deleted ref_file name
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33 cmd1 = 'bowtie-build -f %s %s' % (ref_file_name, ref_file_name ) # bowtie command line, which will work after changing dir (cwd=tmp_index_dir)
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34 try:
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35 FNULL = open(os.devnull, 'w')
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36 tmp = tempfile.NamedTemporaryFile( dir=tmp_index_dir ).name # a path string for a temp file in tmp_index_dir. Just a string
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37 tmp_stderr = open( tmp, 'wb' ) # creates and open a file handler pointing to the temp file
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38 proc = subprocess.Popen( args=cmd1, shell=True, cwd=tmp_index_dir, stderr=FNULL, stdout=FNULL ) # both stderr and stdout of bowtie-build are redirected in dev/null
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39 returncode = proc.wait()
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40 tmp_stderr.close()
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41 FNULL.close()
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42 sys.stdout.write(cmd1 + "\n")
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43 except Exception, e:
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44 # clean up temp dir
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45 if os.path.exists( tmp_index_dir ):
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46 shutil.rmtree( tmp_index_dir )
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47 stop_err( 'Error indexing reference sequence\n' + str( e ) )
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48 # no Cleaning if no Exception, tmp_index_dir has to be cleaned after bowtie_alignment()
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49 index_full_path = os.path.join(tmp_index_dir, ref_file_name) # bowtie fashion path without extention
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50 return index_full_path
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51
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52 def make_working_dir():
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53 working_dir = tempfile.mkdtemp()
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54 return working_dir
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55
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56 def Clean_TempDir(directory):
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57 if os.path.exists( directory ):
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58 shutil.rmtree( directory )
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59 return
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60
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61 def bowtie_alignment(command_line="None", working_dir = ""):
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62 FNULL = open(os.devnull, 'w')
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63 p = subprocess.Popen(args=command_line, cwd=working_dir, shell=True, stderr=FNULL, stdout=FNULL)
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64 returncode = p.wait()
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65 sys.stdout.write("%s\n" % command_line)
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66 FNULL.close()
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67 #p = subprocess.Popen(["wc", "-l", "%s/al.fasta"%working_dir], cwd=working_dir, stdout=subprocess.PIPE)
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68 #aligned = p.communicate()[0].split()[0]
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69 aligned = 0
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70 F = open ("%s/al.fasta" % working_dir, "r")
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71 for line in F:
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72 aligned += 1
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73 F.close()
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74 sys.stdout.write("Aligned: %s\n" % aligned)
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75 return aligned/2
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76
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77 def CommandLiner (v_mis="1", pslots="12", index="dum/my", input="dum/my", working_dir=""):
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78 return "bowtie -v %s -k 1 --best -p %s --al %s/al.fasta --un %s/unal.fasta --suppress 1,2,3,4,5,6,7,8 %s -f %s" % (v_mis, pslots, working_dir, working_dir, index, input)
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79
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80 def __main__():
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81 args = Parser()
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82 ## first we make all indexes available. They can be already available or be squashed by bowtie-build
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83 ## we keep them in a list that alternates indexPath and "toClear" or "DoNotDelete"
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84 BowtieIndexList = []
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85 for indexing_flags, bowtiePath in zip (args.indexing_flags, args.index):
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86 if indexing_flags == "history":
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87 BowtieIndexList.append ( bowtie_squash (bowtiePath) )
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88 BowtieIndexList.append ( "toClear" )
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89 else:
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90 BowtieIndexList.append ( bowtiePath )
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91 BowtieIndexList.append ( "DoNotDelete")
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92 ###### temporary Indexes are generated. They must be deleted at the end (after removing file name in the temp path)
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93 ResultDict = defaultdict(list)
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94 for label, input in zip(args.label, args.input): ## the main cascade, iterating over samples and bowtie indexes
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95 workingDir = make_working_dir()
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96 cmd = CommandLiner (v_mis=args.mismatch, pslots=args.num_threads, index=BowtieIndexList[0], input=input, working_dir=workingDir)
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97 ResultDict[label].append( bowtie_alignment(command_line=cmd, working_dir = workingDir) ) # first step of the cascade
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98 if len(BowtieIndexList) > 2: # is there a second step to perform ?
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99 os.rename("%s/al.fasta"%workingDir, "%s/toAlign.fasta"%workingDir) ## end of first step. the aligned reads are the input of the next step
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100 cmd = CommandLiner (v_mis=args.mismatch, pslots=args.num_threads, index=BowtieIndexList[2], input="%s/toAlign.fasta"%workingDir, working_dir=workingDir)
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101 ResultDict[label].append( bowtie_alignment(command_line=cmd, working_dir = workingDir) )## second step of the cascade
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102 if len(BowtieIndexList) > 4: ## remaining steps
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103 for BowtieIndexPath in BowtieIndexList[4::2]:
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104 os.rename("%s/unal.fasta"%workingDir, "%s/toAlign.fasta"%workingDir)
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105 cmd = CommandLiner (v_mis=args.mismatch, pslots=args.num_threads, index=BowtieIndexPath, input="%s/toAlign.fasta"%workingDir, working_dir=workingDir)
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106 ResultDict[label].append( bowtie_alignment(command_line=cmd, working_dir = workingDir) )
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107 Fun = open("%s/unal.fasta"%workingDir, "r") ## to finish, compute the number of unmatched reads
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108 n = 0
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109 for line in Fun:
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110 n += 1
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111 ResultDict[label].append(n/2)
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112 Fun.close()
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113 Clean_TempDir (workingDir) # clean the sample working directory
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114 ## cleaning
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115 for IndexPath, IndexFlag in zip(BowtieIndexList[::2], BowtieIndexList[1::2]):
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116 if IndexFlag == "toClear":
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117 Clean_TempDir ("/".join(IndexPath.split("/")[:-1]))
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118 ## end of cleaning
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119
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120
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121
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122 F = open (args.output, "w")
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123 print >> F, "alignment reference\t%s" % "\t".join(args.label)
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124 for i, reference in enumerate(args.indexName):
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125 F.write ("%s" % reference)
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126 for sample in args.label:
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127 F.write ("\t%s" % "{:,}".format(ResultDict[sample][i]) )
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128 print >> F
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129 F.write ("Remaining Unmatched")
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130 for sample in args.label:
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131 F.write ("\t%s" % "{:,}".format(ResultDict[sample][-1]) )
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132 print >> F
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133
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134 F.close()
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135
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136 if __name__=="__main__": __main__()
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