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1 #!/usr/bin/env python
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2
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3 import optparse, os, shutil, subprocess, sys, tempfile, fileinput
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4
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5 def stop_err( msg ):
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6 sys.stderr.write( "%s\n" % msg )
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7 sys.exit()
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8
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9 def __main__():
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10 #Parse Command Line
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11 parser = optparse.OptionParser()
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12 parser.add_option( '-p', '--num-threads', dest='num_threads', help='Use this many threads to align reads. The default is 1.' )
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13 parser.add_option( '-C', '--color-space', dest='color_space', action='store_true', help='This indicates color-space data' )
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14 parser.add_option( '-J', '--junctions-output', dest='junctions_output_file', help='Junctions output file; formate is BED.' )
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15 parser.add_option( '-H', '--hits-output', dest='accepted_hits_output_file', help='Accepted hits output file; formate is BAM.' )
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16 parser.add_option( '', '--own-file', dest='own_file', help='' )
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17 parser.add_option( '-D', '--indexes-path', dest='index_path', help='Indexes directory; location of .ebwt and .fa files.' )
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18 parser.add_option( '-r', '--mate-inner-dist', dest='mate_inner_dist', help='This is the expected (mean) inner distance between mate pairs. \
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19 For, example, for paired end runs with fragments selected at 300bp, \
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20 where each end is 50bp, you should set -r to be 200. There is no default, \
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21 and this parameter is required for paired end runs.')
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22 parser.add_option( '', '--mate-std-dev', dest='mate_std_dev', help='Standard deviation of distribution on inner distances between male pairs.' )
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23 parser.add_option( '-a', '--min-anchor-length', dest='min_anchor_length',
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24 help='The "anchor length". TopHat will report junctions spanned by reads with at least this many bases on each side of the junction.' )
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25 parser.add_option( '-m', '--splice-mismatches', dest='splice_mismatches', help='The maximum number of mismatches that can appear in the anchor region of a spliced alignment.' )
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26 parser.add_option( '-i', '--min-intron-length', dest='min_intron_length',
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27 help='The minimum intron length. TopHat will ignore donor/acceptor pairs closer than this many bases apart.' )
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28 parser.add_option( '-I', '--max-intron-length', dest='max_intron_length',
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29 help='The maximum intron length. When searching for junctions ab initio, TopHat will ignore donor/acceptor pairs farther than this many bases apart, except when such a pair is supported by a split segment alignment of a long read.' )
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30 parser.add_option( '-g', '--max_multihits', dest='max_multihits', help='Maximum number of alignments to be allowed' )
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31 parser.add_option( '', '--initial-read-mismatches', dest='initial_read_mismatches', help='Number of mismatches allowed in the initial read mapping' )
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32 parser.add_option( '', '--seg-mismatches', dest='seg_mismatches', help='Number of mismatches allowed in each segment alignment for reads mapped independently' )
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33 parser.add_option( '', '--seg-length', dest='seg_length', help='Minimum length of read segments' )
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34 parser.add_option( '', '--library-type', dest='library_type', help='TopHat will treat the reads as strand specific. Every read alignment will have an XS attribute tag. Consider supplying library type options below to select the correct RNA-seq protocol.' )
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35 parser.add_option( '', '--allow-indels', action="store_true", help='Allow indel search. Indel search is disabled by default.(Not used since version 1.3.0)' )
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36 parser.add_option( '', '--max-insertion-length', dest='max_insertion_length', help='The maximum insertion length. The default is 3.' )
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37 parser.add_option( '', '--max-deletion-length', dest='max_deletion_length', help='The maximum deletion length. The default is 3.' )
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38
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39 # Options for supplying own junctions
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40 parser.add_option( '-G', '--GTF', dest='gene_model_annotations', help='Supply TopHat with a list of gene model annotations. \
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41 TopHat will use the exon records in this file to build \
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42 a set of known splice junctions for each gene, and will \
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43 attempt to align reads to these junctions even if they \
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44 would not normally be covered by the initial mapping.')
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45 parser.add_option( '-j', '--raw-juncs', dest='raw_juncs', help='Supply TopHat with a list of raw junctions. Junctions are \
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46 specified one per line, in a tab-delimited format. Records \
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47 look like: <chrom> <left> <right> <+/-> left and right are \
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48 zero-based coordinates, and specify the last character of the \
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49 left sequenced to be spliced to the first character of the right \
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50 sequence, inclusive.')
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51 parser.add_option( '', '--no-novel-juncs', action="store_true", dest='no_novel_juncs', help="Only look for junctions indicated in the \
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52 supplied GFF file. (ignored without -G)")
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53 parser.add_option( '', '--no-novel-indels', action="store_true", dest='no_novel_indels', help="Skip indel search. Indel search is enabled by default.")
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54 # Types of search.
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55 parser.add_option( '', '--microexon-search', action="store_true", dest='microexon_search', help='With this option, the pipeline will attempt to find alignments incident to microexons. Works only for reads 50bp or longer.')
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56 parser.add_option( '', '--closure-search', action="store_true", dest='closure_search', help='Enables the mate pair closure-based search for junctions. Closure-based search should only be used when the expected inner distance between mates is small (<= 50bp)')
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57 parser.add_option( '', '--no-closure-search', action="store_false", dest='closure_search' )
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58 parser.add_option( '', '--coverage-search', action="store_true", dest='coverage_search', help='Enables the coverage based search for junctions. Use when coverage search is disabled by default (such as for reads 75bp or longer), for maximum sensitivity.')
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59 parser.add_option( '', '--no-coverage-search', action="store_false", dest='coverage_search' )
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60 parser.add_option( '', '--min-segment-intron', dest='min_segment_intron', help='Minimum intron length that may be found during split-segment search' )
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61 parser.add_option( '', '--max-segment-intron', dest='max_segment_intron', help='Maximum intron length that may be found during split-segment search' )
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62 parser.add_option( '', '--min-closure-exon', dest='min_closure_exon', help='Minimum length for exonic hops in potential splice graph' )
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63 parser.add_option( '', '--min-closure-intron', dest='min_closure_intron', help='Minimum intron length that may be found during closure search' )
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64 parser.add_option( '', '--max-closure-intron', dest='max_closure_intron', help='Maximum intron length that may be found during closure search' )
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65 parser.add_option( '', '--min-coverage-intron', dest='min_coverage_intron', help='Minimum intron length that may be found during coverage search' )
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66 parser.add_option( '', '--max-coverage-intron', dest='max_coverage_intron', help='Maximum intron length that may be found during coverage search' )
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67
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68 # Wrapper options.
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69 parser.add_option( '-1', '--input1', dest='input1', help='The (forward or single-end) reads file in Sanger FASTQ format' )
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70 parser.add_option( '-2', '--input2', dest='input2', help='The reverse reads file in Sanger FASTQ format' )
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71 parser.add_option( '', '--single-paired', dest='single_paired', help='' )
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72 parser.add_option( '', '--settings', dest='settings', help='' )
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73
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74 (options, args) = parser.parse_args()
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75
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76 # output version # of tool
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77 try:
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78 tmp = tempfile.NamedTemporaryFile().name
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79 tmp_stdout = open( tmp, 'wb' )
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80 proc = subprocess.Popen( args='tophat -v', shell=True, stdout=tmp_stdout )
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81 tmp_stdout.close()
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82 returncode = proc.wait()
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83 stdout = open( tmp_stdout.name, 'rb' ).readline().strip()
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84 if stdout:
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85 sys.stdout.write( '%s\n' % stdout )
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86 else:
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87 raise Exception
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88 except:
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89 sys.stdout.write( 'Could not determine Tophat version\n' )
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90
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91 # Color or base space
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92 space = ''
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93 if options.color_space:
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94 space = '-C'
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95
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96 # Creat bowtie index if necessary.
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97 tmp_index_dir = tempfile.mkdtemp()
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98 if options.own_file:
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99 index_path = os.path.join( tmp_index_dir, '.'.join( os.path.split( options.own_file )[1].split( '.' )[:-1] ) )
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100 try:
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101 os.link( options.own_file, index_path + '.fa' )
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102 except:
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103 # Tophat prefers (but doesn't require) fasta file to be in same directory, with .fa extension
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104 pass
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105 cmd_index = 'bowtie-build %s -f %s %s' % ( space, options.own_file, index_path )
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106 try:
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107 tmp = tempfile.NamedTemporaryFile( dir=tmp_index_dir ).name
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108 tmp_stderr = open( tmp, 'wb' )
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109 proc = subprocess.Popen( args=cmd_index, shell=True, cwd=tmp_index_dir, stderr=tmp_stderr.fileno() )
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110 returncode = proc.wait()
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111 tmp_stderr.close()
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112 # get stderr, allowing for case where it's very large
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113 tmp_stderr = open( tmp, 'rb' )
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114 stderr = ''
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115 buffsize = 1048576
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116 try:
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117 while True:
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118 stderr += tmp_stderr.read( buffsize )
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119 if not stderr or len( stderr ) % buffsize != 0:
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120 break
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121 except OverflowError:
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122 pass
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123 tmp_stderr.close()
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124 if returncode != 0:
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125 raise Exception, stderr
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126 except Exception, e:
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127 if os.path.exists( tmp_index_dir ):
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128 shutil.rmtree( tmp_index_dir )
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129 stop_err( 'Error indexing reference sequence\n' + str( e ) )
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130 else:
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131 index_path = options.index_path
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132
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133 # Build tophat command.
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134 cmd = 'tophat %s %s %s'
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135 reads = options.input1
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136 if options.input2:
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137 reads += ' ' + options.input2
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138 opts = '-p %s %s' % ( options.num_threads, space )
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139 if options.single_paired == 'paired':
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140 opts += ' -r %s' % options.mate_inner_dist
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141 if options.settings == 'preSet':
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142 cmd = cmd % ( opts, index_path, reads )
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143 else:
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144 try:
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145 if int( options.min_anchor_length ) >= 3:
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146 opts += ' -a %s' % options.min_anchor_length
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147 else:
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148 raise Exception, 'Minimum anchor length must be 3 or greater'
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149 opts += ' -m %s' % options.splice_mismatches
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150 opts += ' -i %s' % options.min_intron_length
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151 opts += ' -I %s' % options.max_intron_length
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152 opts += ' -g %s' % options.max_multihits
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153 # Custom junctions options.
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154 if options.gene_model_annotations:
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155 opts += ' -G %s' % options.gene_model_annotations
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156 if options.raw_juncs:
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157 opts += ' -j %s' % options.raw_juncs
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158 if options.no_novel_juncs:
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159 opts += ' --no-novel-juncs'
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160 if options.library_type:
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161 opts += ' --library-type %s' % options.library_type
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162 if options.no_novel_indels:
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163 opts += ' --no-novel-indels'
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164 else:
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165 if options.max_insertion_length:
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166 opts += ' --max-insertion-length %i' % int( options.max_insertion_length )
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167 if options.max_deletion_length:
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168 opts += ' --max-deletion-length %i' % int( options.max_deletion_length )
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169 # Max options do not work for Tophat v1.2.0, despite documentation to the contrary. (Fixed in version 1.3.1)
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170 # need to warn user of this fact
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171 #sys.stdout.write( "Max insertion length and max deletion length options don't work in Tophat v1.2.0\n" )
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172
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173 # Search type options.
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174 if options.coverage_search:
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175 opts += ' --coverage-search --min-coverage-intron %s --max-coverage-intron %s' % ( options.min_coverage_intron, options.max_coverage_intron )
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176 else:
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177 opts += ' --no-coverage-search'
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178 if options.closure_search:
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179 opts += ' --closure-search --min-closure-exon %s --min-closure-intron %s --max-closure-intron %s' % ( options.min_closure_exon, options.min_closure_intron, options.max_closure_intron )
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180 else:
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181 opts += ' --no-closure-search'
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182 if options.microexon_search:
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183 opts += ' --microexon-search'
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184 if options.single_paired == 'paired':
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185 opts += ' --mate-std-dev %s' % options.mate_std_dev
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186 if options.initial_read_mismatches:
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187 opts += ' --initial-read-mismatches %d' % int( options.initial_read_mismatches )
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188 if options.seg_mismatches:
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189 opts += ' --segment-mismatches %d' % int( options.seg_mismatches )
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190 if options.seg_length:
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191 opts += ' --segment-length %d' % int( options.seg_length )
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192 if options.min_segment_intron:
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193 opts += ' --min-segment-intron %d' % int( options.min_segment_intron )
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194 if options.max_segment_intron:
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195 opts += ' --max-segment-intron %d' % int( options.max_segment_intron )
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196 cmd = cmd % ( opts, index_path, reads )
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197 except Exception, e:
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198 # Clean up temp dirs
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199 if os.path.exists( tmp_index_dir ):
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200 shutil.rmtree( tmp_index_dir )
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201 stop_err( 'Something is wrong with the alignment parameters and the alignment could not be run\n' + str( e ) )
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202 #print cmd
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203
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204 # Run
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205 try:
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206 tmp_out = tempfile.NamedTemporaryFile().name
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207 tmp_stdout = open( tmp_out, 'wb' )
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208 tmp_err = tempfile.NamedTemporaryFile().name
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209 tmp_stderr = open( tmp_err, 'wb' )
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210 print cmd
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211 proc = subprocess.Popen( args=cmd, shell=True, cwd=".", stdout=tmp_stdout, stderr=tmp_stderr )
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212 returncode = proc.wait()
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213 tmp_stderr.close()
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214 # get stderr, allowing for case where it's very large
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215 tmp_stderr = open( tmp_err, 'rb' )
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216 stderr = ''
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217 buffsize = 1048576
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218 try:
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219 while True:
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220 stderr += tmp_stderr.read( buffsize )
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221 if not stderr or len( stderr ) % buffsize != 0:
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222 break
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223 except OverflowError:
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224 pass
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225 tmp_stdout.close()
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226 tmp_stderr.close()
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227 if returncode != 0:
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228 raise Exception, stderr
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229
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230 # TODO: look for errors in program output.
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231 except Exception, e:
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232 stop_err( 'Error in tophat:\n' + str( e ) )
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233
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234 # Clean up temp dirs
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235 if os.path.exists( tmp_index_dir ):
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236 shutil.rmtree( tmp_index_dir )
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237
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238 if __name__=="__main__": __main__()
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