#!/usr/bin/env python import optparse, os, shutil, subprocess, sys, tempfile, fileinput def stop_err( msg ): sys.stderr.write( "%s\n" % msg ) sys.exit() def __main__(): #Parse Command Line parser = optparse.OptionParser() parser.add_option( '-p', '--num-threads', dest='num_threads', help='Use this many threads to align reads. The default is 1.' ) parser.add_option( '-C', '--coverage-output', dest='coverage_output_file', help='Coverage output file; formate is WIG.' ) parser.add_option( '-J', '--junctions-output', dest='junctions_output_file', help='Junctions output file; formate is BED.' ) parser.add_option( '-H', '--hits-output', dest='accepted_hits_output_file', help='Accepted hits output file; formate is SAM.' ) parser.add_option( '', '--own-file', dest='own_file', help='' ) parser.add_option( '-D', '--indexes-path', dest='index_path', help='Indexes directory; location of .ebwt and .fa files.' ) parser.add_option( '-r', '--mate-inner-dist', dest='mate_inner_dist', help='This is the expected (mean) inner distance between mate pairs. \ For, example, for paired end runs with fragments selected at 300bp, \ where each end is 50bp, you should set -r to be 200. There is no default, \ and this parameter is required for paired end runs.') parser.add_option( '', '--mate-std-dev', dest='mate_std_dev', help='Standard deviation of distribution on inner distances between male pairs.' ) parser.add_option( '-a', '--min-anchor-length', dest='min_anchor_length', help='The "anchor length". TopHat will report junctions spanned by reads with at least this many bases on each side of the junction.' ) 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.' ) parser.add_option( '-i', '--min-intron-length', dest='min_intron_length', help='The minimum intron length. TopHat will ignore donor/acceptor pairs closer than this many bases apart.' ) parser.add_option( '-I', '--max-intron-length', dest='max_intron_length', 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.' ) parser.add_option( '-F', '--junction_filter', dest='junction_filter', help='Filter out junctions supported by too few alignments (number of reads divided by average depth of coverage)' ) parser.add_option( '-g', '--max_multihits', dest='max_multihits', help='Maximum number of alignments to be allowed' ) 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.') 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)') parser.add_option( '', '--no-closure-search', action="store_false", dest='closure_search' ) 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.') parser.add_option( '', '--no-coverage-search', action="store_false", dest='coverage_search' ) parser.add_option( '', '--min-segment-intron', dest='min_segment_intron', help='Minimum intron length that may be found during split-segment search' ) parser.add_option( '', '--max-segment-intron', dest='max_segment_intron', help='Maximum intron length that may be found during split-segment search' ) parser.add_option( '', '--min-closure-exon', dest='min_closure_exon', help='Minimum length for exonic hops in potential splice graph' ) parser.add_option( '', '--min-closure-intron', dest='min_closure_intron', help='Minimum intron length that may be found during closure search' ) parser.add_option( '', '--max-closure-intron', dest='max_closure_intron', help='Maximum intron length that may be found during closure search' ) parser.add_option( '', '--min-coverage-intron', dest='min_coverage_intron', help='Minimum intron length that may be found during coverage search' ) parser.add_option( '', '--max-coverage-intron', dest='max_coverage_intron', help='Maximum intron length that may be found during coverage search' ) parser.add_option( '', '--seg-mismatches', dest='seg_mismatches', help='Number of mismatches allowed in each segment alignment for reads mapped independently' ) parser.add_option( '', '--seg-length', dest='seg_length', help='Minimum length of read segments' ) # Wrapper options. parser.add_option( '-1', '--input1', dest='input1', help='The (forward or single-end) reads file in Sanger FASTQ format' ) parser.add_option( '-2', '--input2', dest='input2', help='The reverse reads file in Sanger FASTQ format' ) parser.add_option( '', '--single-paired', dest='single_paired', help='' ) parser.add_option( '', '--settings', dest='settings', help='' ) (options, args) = parser.parse_args() #sys.stderr.write('*'*50+'\n'+str(options)+'\n'+'*'*50+'\n') # Creat bowtie index if necessary. tmp_index_dir = tempfile.mkdtemp() if options.own_file != 'None': index_path = os.path.join( tmp_index_dir, os.path.split( options.own_file )[1] ) cmd_index = 'bowtie-build -f %s %s' % ( options.own_file, index_path ) try: tmp = tempfile.NamedTemporaryFile( dir=tmp_index_dir ).name tmp_stderr = open( tmp, 'wb' ) proc = subprocess.Popen( args=cmd_index, shell=True, cwd=tmp_index_dir, stderr=tmp_stderr.fileno() ) returncode = proc.wait() tmp_stderr.close() # get stderr, allowing for case where it's very large tmp_stderr = open( tmp, 'rb' ) stderr = '' buffsize = 1048576 try: while True: stderr += tmp_stderr.read( buffsize ) if not stderr or len( stderr ) % buffsize != 0: break except OverflowError: pass tmp_stderr.close() if returncode != 0: raise Exception, stderr except Exception, e: if os.path.exists( tmp_index_dir ): shutil.rmtree( tmp_index_dir ) stop_err( 'Error indexing reference sequence\n' + str( e ) ) else: index_path = options.index_path # Build tophat command. tmp_output_dir = tempfile.mkdtemp() cmd = 'tophat -o %s %s %s %s' reads = options.input1 if options.input2 != 'None': reads += ' ' + options.input2 opts = '-p %s' % options.num_threads if options.single_paired == 'paired': opts += ' -r %s' % options.mate_inner_dist if options.settings == 'preSet': cmd = cmd % ( tmp_output_dir, opts, index_path, reads ) else: try: if int( options.min_anchor_length ) >= 3: opts += ' -a %s' % options.min_anchor_length else: raise Exception, 'Minimum anchor length must be 3 or greater' opts += ' -m %s' % options.splice_mismatches opts += ' -i %s' % options.min_intron_length opts += ' -I %s' % options.max_intron_length if float( options.junction_filter ) != 0.0: opts += ' -F %s' % options.junction_filter opts += ' -g %s' % options.max_multihits if options.coverage_search: opts += ' --coverage-search --min-coverage-intron %s --max-coverage-intron %s' % ( options.min_coverage_intron, options.max_coverage_intron ) else: opts += ' --no-coverage-search' if options.closure_search: 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 ) else: opts += ' --no-closure-search' if options.microexon_search: opts += ' --microexon-search' if options.single_paired == 'paired': opts += ' --mate-std-dev %s' % options.mate_std_dev cmd = cmd % ( tmp_output_dir, opts, index_path, reads ) except Exception, e: # Clean up temp dirs if os.path.exists( tmp_index_dir ): shutil.rmtree( tmp_index_dir ) if os.path.exists( tmp_output_dir ): shutil.rmtree( tmp_output_dir ) stop_err( 'Something is wrong with the alignment parameters and the alignment could not be run\n' + str( e ) ) print cmd # Run try: tmp_out = tempfile.NamedTemporaryFile( dir=tmp_output_dir ).name tmp_stdout = open( tmp_out, 'wb' ) tmp_err = tempfile.NamedTemporaryFile( dir=tmp_output_dir ).name tmp_stderr = open( tmp_err, 'wb' ) proc = subprocess.Popen( args=cmd, shell=True, cwd=tmp_output_dir, stdout=tmp_stdout, stderr=tmp_stderr ) returncode = proc.wait() tmp_stderr.close() # get stderr, allowing for case where it's very large tmp_stderr = open( tmp_err, 'rb' ) stderr = '' buffsize = 1048576 try: while True: stderr += tmp_stderr.read( buffsize ) if not stderr or len( stderr ) % buffsize != 0: break except OverflowError: pass tmp_stdout.close() tmp_stderr.close() if returncode != 0: raise Exception, stderr except Exception, e: # Clean up temp dirs if os.path.exists( tmp_output_dir ): shutil.rmtree( tmp_output_dir ) stop_err( 'Error in tophat:\n' + str( e ) ) # TODO: look for errors in program output. # Postprocessing: copy output files from tmp directory to specified files. Also need to remove header lines from SAM file. try: try: shutil.copyfile( tmp_output_dir + "/coverage.wig", options.coverage_output_file ) shutil.copyfile( tmp_output_dir + "/junctions.bed", options.junctions_output_file ) # Remove headers from SAM file in place. in_header = True # Headers always at start of file. for line in fileinput.input( tmp_output_dir + "/accepted_hits.sam", inplace=1 ): if in_header and line.startswith("@"): continue else: in_header = False sys.stdout.write( line ) # Copy SAM File. shutil.copyfile( tmp_output_dir + "/accepted_hits.sam", options.accepted_hits_output_file ) except Exception, e: stop_err( 'Error in tophat:\n' + str( e ) ) finally: # Clean up temp dirs if os.path.exists( tmp_index_dir ): shutil.rmtree( tmp_index_dir ) if os.path.exists( tmp_output_dir ): shutil.rmtree( tmp_output_dir ) if __name__=="__main__": __main__()