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56
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1 # May 2013
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2 # Change to htseq as the counting engine - wrap so arbitrary number of columns created
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3 # borged Simon Anders' "count.py" since we need a vector of counts rather than a new sam file as output
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4 # note attribution for htseq and count.py :
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5 ## Written by Simon Anders (sanders@fs.tum.de), European Molecular Biology
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6 ## Laboratory (EMBL). (c) 2010. Released under the terms of the GNU General
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7 ## Public License v3. Part of the 'HTSeq' framework, version HTSeq-0.5.4p3
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8 # updated ross lazarus august 2011 to NOT include region and to finesse the name as the region for bed3 format inputs
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9 # also now sums all duplicate named regions and provides a summary of any collapsing as the info
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10 # updated ross lazarus july 26 to respect the is_duplicate flag rather than try to second guess
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11 # note Heng Li argues that removing dupes is a bad idea for RNA seq
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12 # updated ross lazarus july 22 to count reads OUTSIDE each bed region during the processing of each bam
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13 # added better sorting with decoration of a dict key later sorted and undecorated.
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14 # code cleaned up and galaxified ross lazarus july 18 et seq.
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15 # bams2mx.py -turns a series of bam and a bed file into a matrix of counts Usage bams2mx.py <halfwindow> <bedfile.bed> <bam1.bam>
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16 # <bam2.bam>
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17 # uses pysam to read and count bam reads over each bed interval for each sample for speed
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18 # still not so fast
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19 # TODO options -shift -unique
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20 #
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21 """
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22 how this gets run:
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23
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24 (vgalaxy)galaxy@iaas1-int:~$ cat database/job_working_directory/027/27014/galaxy_27014.sh
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25 #!/bin/sh
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26 GALAXY_LIB="/data/extended/galaxy/lib"
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27 if [ "$GALAXY_LIB" != "None" ]; then
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28 if [ -n "$PYTHONPATH" ]; then
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29 PYTHONPATH="$GALAXY_LIB:$PYTHONPATH"
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30 else
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31 PYTHONPATH="$GALAXY_LIB"
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32 fi
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33 export PYTHONPATH
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34 fi
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35
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36 cd /data/extended/galaxy/database/job_working_directory/027/27014
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37 python /data/extended/galaxy/tools/rgenetics/htseqsams2mx.py -g "/data/extended/galaxy/database/files/034/dataset_34115.dat" -o "/data/extended/galaxy/database/files/034/dataset_34124.dat" -m "union" --id_attribute "gene_id" --feature_type "exon" --samf "'/data/extended/galaxy/database/files/033/dataset_33980.dat','T5A_C1PPHACXX_AGTTCC_L003_R1.fastq_bwa.sam'" --samf "'/data/extended/galaxy/database/files/033/dataset_33975.dat','T5A_C1PPHACXX_AGTTCC_L002_R1.fastq_bwa.sam'"; cd /data/extended/galaxy; /data/extended/galaxy/set_metadata.sh ./database/files /data/extended/galaxy/database/job_working_directory/027/27014 . /data/extended/galaxy/universe_wsgi.ini /data/tmp/tmpmwsElH /data/extended/galaxy/database/job_working_directory/027/27014/galaxy.json /data/extended/galaxy/database/job_working_directory/027/27014/metadata_in_HistoryDatasetAssociation_45202_sfOMGa,/data/extended/galaxy/database/job_working_directory/027/27014/metadata_kwds_HistoryDatasetAssociation_45202_gaMnxa,/data/extended/galaxy/database/job_working_directory/027/27014/metadata_out_HistoryDatasetAssociation_45202_kZPsZO,/data/extended/galaxy/database/job_working_directory/027/27014/metadata_results_HistoryDatasetAssociation_45202_bXU7IU,,/data/extended/galaxy/database/job_working_directory/027/27014/metadata_override_HistoryDatasetAssociation_45202_hyLAvh
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38 echo $? > /data/extended/galaxy/database/job_working_directory/027/27014/galaxy_27014.ec
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39
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40 """
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41
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42 import os
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43 import re
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44 import sys
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45 import HTSeq.scripts.count as htcount
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46 import optparse
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47 import tempfile
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48 import shutil
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49 import operator
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50 import subprocess
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51 import itertools
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52 import warnings
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53 import traceback
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54 import HTSeq
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55 import time
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56
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57
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58 class Xcpt(Exception):
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59 def __init__(self, msg):
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60 self.msg = msg
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61
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62
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63 def htseqMX(gff_filename,sam_filenames,colnames,sam_exts,sam_bais,opts):
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64 """
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65 Code taken from count.py in Simon Anders HTSeq distribution
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66 Wrapped in a loop to accept multiple bam/sam files and their names from galaxy to
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67 produce a matrix of contig counts by sample for downstream use in edgeR and DESeq tools
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68 """
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69 class UnknownChrom( Exception ):
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70 pass
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71
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72 def my_showwarning( message, category, filename, lineno = None, line = None ):
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73 sys.stdout.write( "Warning: %s\n" % message )
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74
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75 def invert_strand( iv ):
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76 iv2 = iv.copy()
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77 if iv2.strand == "+":
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78 iv2.strand = "-"
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79 elif iv2.strand == "-":
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80 iv2.strand = "+"
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81 else:
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82 raise ValueError, "Illegal strand"
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83 return iv2
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84
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85 def count_reads_in_features( sam_filenames, colnames, gff_filename, opts ):
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86 """ Hacked version of htseq count.py
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87 """
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88 if opts.quiet:
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89 warnings.filterwarnings( action="ignore", module="HTSeq" )
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90 features = HTSeq.GenomicArrayOfSets( "auto", opts.stranded != "no" )
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91 mapqMin = int(opts.mapqMin)
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92 counts = {}
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93 nreads = 0
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94 empty = 0
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95 ambiguous = 0
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96 notaligned = 0
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97 lowqual = 0
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98 nonunique = 0
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99 filtered = 0 # new filter_extras - need a better way to do this - independent filter tool?
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100 gff = HTSeq.GFF_Reader( gff_filename )
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101 try:
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102 for i,f in enumerate(gff):
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103 if f.type == opts.feature_type:
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104 try:
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105 feature_id = f.attr[ opts.id_attribute ]
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106 except KeyError:
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107 try:
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108 feature_id = f.attr[ 'gene_id' ]
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109 except KeyError:
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110 sys.exit( "Feature at row %d %s does not contain a '%s' attribute OR a gene_id attribute - faulty GFF?" %
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111 ( (i+1), f.name, opts.id_attribute ) )
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112 if opts.stranded != "no" and f.iv.strand == ".":
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113 sys.exit( "Feature %s at %s does not have strand information but you are "
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114 "running htseq-count in stranded mode. Use '--stranded=no'." %
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115 ( f.name, f.iv ) )
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116 features[ f.iv ] += feature_id
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117 counts[ feature_id ] = [0 for x in colnames] # we use sami as an index here to bump counts later
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118 except:
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119 sys.stderr.write( "Error occured in %s.\n" % gff.get_line_number_string() )
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120 raise
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121
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122 if not opts.quiet:
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123 sys.stdout.write( "%d GFF lines processed.\n" % i )
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124
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125 if len( counts ) == 0 and not opts.quiet:
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126 sys.stdout.write( "Warning: No features of type '%s' found.\n" % opts.feature_type )
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127 for sami,sam_filename in enumerate(sam_filenames):
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128 colname = colnames[sami]
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129 isbam = sam_exts[sami] == 'bam'
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130 hasbai = sam_bais[sami] > ''
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131 if hasbai:
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132 tempname = os.path.splitext(os.path.basename(sam_filename))[0]
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133 tempbam = '%s_TEMP.bam' % tempname
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134 tempbai = '%s_TEMP.bai' % tempname
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135 os.link(sam_filename,tempbam)
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136 os.link(sam_bais[sami],tempbai)
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137 try:
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138 if isbam:
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139 if hasbai:
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140 read_seq = HTSeq.BAM_Reader ( tempbam )
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141 else:
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142 read_seq = HTSeq.BAM_Reader( sam_filename )
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143 else:
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144 read_seq = HTSeq.SAM_Reader( sam_filename )
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145 first_read = iter(read_seq).next()
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146 pe_mode = first_read.paired_end
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147 except:
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148 if isbam:
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149 print >> sys.stderr, "Error occured when reading first line of bam file %s colname=%s \n" % (sam_filename,colname )
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150 else:
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151 print >> sys.stderr, "Error occured when reading first line of sam file %s colname=%s \n" % (sam_filename,colname )
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152 raise
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153
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154 try:
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155 if pe_mode:
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156 read_seq_pe_file = read_seq
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157 read_seq = HTSeq.pair_SAM_alignments( read_seq )
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158 for seqi,r in enumerate(read_seq):
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159 nreads += 1
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160 if not pe_mode:
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161 if not r.aligned:
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162 notaligned += 1
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163 continue
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164 try:
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165 if len(opts.filter_extras) > 0:
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166 for extra in opts.filter_extras:
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167 if r.optional_field(extra):
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168 filtered += 1
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169 continue
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170 if r.optional_field( "NH" ) > 1:
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171 nonunique += 1
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172 continue
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173 except KeyError:
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174 pass
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175 if r.aQual < mapqMin:
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176 lowqual += 1
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177 continue
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178 if opts.stranded != "reverse":
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179 iv_seq = ( co.ref_iv for co in r.cigar if co.type == "M" and co.size > 0 )
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180 else:
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181 iv_seq = ( invert_strand( co.ref_iv ) for co in r.cigar if co.type == "M" and co.size > 0 )
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182 else:
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183 if r[0] is not None and r[0].aligned:
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184 if opts.stranded != "reverse":
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185 iv_seq = ( co.ref_iv for co in r[0].cigar if co.type == "M" and co.size > 0 )
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186 else:
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187 iv_seq = ( invert_strand( co.ref_iv ) for co in r[0].cigar if co.type == "M" and co.size > 0 )
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188 else:
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189 iv_seq = tuple()
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190 if r[1] is not None and r[1].aligned:
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191 if opts.stranded != "reverse":
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192 iv_seq = itertools.chain( iv_seq,
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193 ( invert_strand( co.ref_iv ) for co in r[1].cigar if co.type == "M" and co.size > 0 ) )
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194 else:
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195 iv_seq = itertools.chain( iv_seq,
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196 ( co.ref_iv for co in r[1].cigar if co.type == "M" and co.size > 0 ) )
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197 else:
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198 if ( r[0] is None ) or not ( r[0].aligned ):
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199 notaligned += 1
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200 continue
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201 try:
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202 if ( r[0] is not None and r[0].optional_field( "NH" ) > 1 ) or \
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203 ( r[1] is not None and r[1].optional_field( "NH" ) > 1 ):
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204 nonunique += 1
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205 continue
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206 except KeyError:
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207 pass
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208 if ( r[0] and r[0].aQual < mapqMin ) or ( r[1] and r[1].aQual < mapqMin ):
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209 lowqual += 1
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210 continue
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211
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212 try:
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213 if opts.mode == "union":
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214 fs = set()
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215 for iv in iv_seq:
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216 if iv.chrom not in features.chrom_vectors:
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217 raise UnknownChrom
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218 for iv2, fs2 in features[ iv ].steps():
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219 fs = fs.union( fs2 )
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220 elif opts.mode == "intersection-strict" or opts.mode == "intersection-nonempty":
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221 fs = None
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222 for iv in iv_seq:
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223 if iv.chrom not in features.chrom_vectors:
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224 raise UnknownChrom
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225 for iv2, fs2 in features[ iv ].steps():
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226 if len(fs2) > 0 or opts.mode == "intersection-strict":
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227 if fs is None:
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228 fs = fs2.copy()
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229 else:
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230 fs = fs.intersection( fs2 )
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231 else:
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232 sys.exit( "Illegal overlap mode %s" % opts.mode )
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233 if fs is None or len( fs ) == 0:
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234 empty += 1
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235 elif len( fs ) > 1:
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236 ambiguous += 1
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237 else:
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238 ck = list(fs)[0]
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239 counts[ck][sami] += 1 # end up with counts for each sample as a list
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240 except UnknownChrom:
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241 if not pe_mode:
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242 rr = r
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243 else:
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244 rr = r[0] if r[0] is not None else r[1]
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245 empty += 1
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246 if not opts.quiet:
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247 sys.stdout.write( ( "Warning: Skipping read '%s', because chromosome " +
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248 "'%s', to which it has been aligned, did not appear in the GFF file.\n" ) %
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249 ( rr.read.name, iv.chrom ) )
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250 except:
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251 if not pe_mode:
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252 sys.stderr.write( "Error occured in %s.\n" % read_seq.get_line_number_string() )
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253 else:
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254 sys.stderr.write( "Error occured in %s.\n" % read_seq_pe_file.get_line_number_string() )
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255 raise
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256
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257 if not opts.quiet:
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258 sys.stdout.write( "%d sam %s processed for %s.\n" % ( seqi, "lines " if not pe_mode else "line pairs", colname ) )
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259 return counts,empty,ambiguous,lowqual,notaligned,nonunique,filtered,nreads
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260
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261 warnings.showwarning = my_showwarning
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262 assert os.path.isfile(gff_filename),'## unable to open supplied gff file %s' % gff_filename
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263 try:
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264 counts,empty,ambiguous,lowqual,notaligned,nonunique,filtered,nreads = count_reads_in_features( sam_filenames, colnames, gff_filename,opts)
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265 except:
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266 sys.stderr.write( "Error: %s\n" % str( sys.exc_info()[1] ) )
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267 sys.stderr.write( "[Exception type: %s, raised in %s:%d]\n" %
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268 ( sys.exc_info()[1].__class__.__name__,
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269 os.path.basename(traceback.extract_tb( sys.exc_info()[2] )[-1][0]),
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270 traceback.extract_tb( sys.exc_info()[2] )[-1][1] ) )
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271 sys.exit( 1 )
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272 return counts,empty,ambiguous,lowqual,notaligned,nonunique,filtered,nreads
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273
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274
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275 def usage():
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276 print >> sys.stdout, """Usage: python htseqsams2mx.py -w <halfwindowsize> -g <gfffile.gff> -o <outfilename> [-i] [-c] --samf "<sam1.sam>,<sam1.column_header>" --samf "...<samN.column_header>" """
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277 sys.exit(1)
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278
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279 if __name__ == "__main__":
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280 """
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281 <command interpreter="python">
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282 htseqsams2mx.py -w "$halfwin" -g "$gfffile" -o "$outfile" -m "union"
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283 #for $s in $samfiles:
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284 --samf "'${s.samf}','${s.samf.name}'"
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285 #end for
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286 </command>
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287 """
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288 if len(sys.argv) < 2:
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289 usage()
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290 sys.exit(1)
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291 starttime = time.time()
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292 op = optparse.OptionParser()
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293 # All tools
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294 op.add_option('-w', '--halfwindow', default="0")
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295 op.add_option('-m', '--mode', default="union")
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296 op.add_option('-s', '--stranded', default="no")
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297 op.add_option('-y', '--feature_type', default="exon")
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298 op.add_option('-g', '--gff_file', default=None)
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299 op.add_option('-o', '--outfname', default=None)
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300 op.add_option('-f','--forceName', default="false")
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301 op.add_option('--samf', default=[], action="append")
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302 op.add_option('--filter_extras', default=[], action="append")
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303 op.add_option('--mapqMin', default='0')
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304 op.add_option( "-t", "--type", type="string", dest="featuretype",
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305 default = "exon", help = "feature type (3rd column in GFF file) to be used, " +
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306 "all features of other type are ignored (default, suitable for Ensembl " +
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307 "GTF files: exon)" )
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308
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309 op.add_option( "-i", "--id_attribute", type="string", dest="id_attribute",
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310 default = "gene_name", help = "GTF attribute to be used as feature ID (default, " +
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311 "suitable for Ensembl GTF files: gene_id)" )
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312
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313 op.add_option( "-q", "--quiet", action="store_true", dest="quiet", default = False,
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314 help = "suppress progress report and warnings" )
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315 opts, args = op.parse_args()
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316 halfwindow = int(opts.halfwindow)
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317 gff_file = opts.gff_file
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318 assert os.path.isfile(gff_file),'##ERROR htseqsams2mx: Supplied input GFF file "%s" not found' % gff_file
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319 outfname = opts.outfname
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320 sam_filenames = []
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321 colnames = []
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322 samf = opts.samf
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323 samfsplit = [x.split(',') for x in samf] # one per samf set
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324 samsets = []
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325 for samfs in samfsplit:
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326 samset = [x.replace("'","") for x in samfs]
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327 samset = [x.replace('"','') for x in samset]
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328 samsets.append(samset)
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329 samsets = [x for x in samsets if x[0].lower() != 'none']
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330 # just cannot stop getting these on cl! wtf in cheetah for a repeat group?
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331 samfnames = [x[0] for x in samsets]
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332 if len(set(samfnames)) != len(samfnames):
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333 samnames = []
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334 delme = []
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335 for i,s in enumerate(samfnames):
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336 if s in samnames:
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337 delme.append(i)
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338 print sys.stdout,'## WARNING htseqsams2mx: Duplicate input sam file %s in %s - ignoring dupe in 0 based position %s' %\
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339 (s,','.join(samfnames), str(delme))
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340 else:
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341 samnames.append(s) # first time
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342 samsets = [x for i,x in enumerate(samsets) if not (i in delme)]
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343 samfnames = [x[0] for x in samsets]
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344 scolnames = [x[1]for x in samsets]
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345 assert len(samfnames) == len(scolnames), '##ERROR sams2mx: Count of sam/cname not consistent - %d/%d' % (len(samfnames),len(scolnames))
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346 sam_exts = [x[2] for x in samsets]
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347 assert len(samfnames) == len(sam_exts), '##ERROR sams2mx: Count of extensions not consistent - %d/%d' % (len(samfnames),len(sam_exts))
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348 sam_bais = [x[3] for x in samsets] # these only exist for bams and need to be finessed with a symlink so pysam will just work
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349 for i,b in enumerate(samfnames):
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350 assert os.path.isfile(b),'## Supplied input sam file "%s" not found' % b
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351 sam_filenames.append(b)
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352 sampName = scolnames[i] # better be unique
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353 sampName = sampName.replace('#','') # for R
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354 sampName = sampName.replace('(','') # for R
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355 sampName = sampName.replace(')','') # for R
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356 sampName = sampName.replace(' ','_') # for R
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357 colnames.append(sampName)
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358 counts,empty,ambiguous,lowqual,notaligned,nonunique,filtered,nreads = htseqMX(gff_file, sam_filenames,colnames,sam_exts,sam_bais,opts)
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359 heads = '\t'.join(['Contig',] + colnames)
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360 res = [heads,]
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361 contigs = counts.keys()
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362 contigs.sort()
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363 totalc = 0
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364 emptycontigs = 0
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365 for contig in contigs:
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366 thisc = sum(counts[contig])
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367 if thisc > 0: # no output for empty contigs
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368 totalc += thisc
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369 crow = [contig,] + ['%d' % x for x in counts[contig]]
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370 res.append('\t'.join(crow))
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371 else:
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372 emptycontigs += 1
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373 outf = open(opts.outfname,'w')
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374 outf.write('\n'.join(res))
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375 outf.write('\n')
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376 outf.close()
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377 walltime = int(time.time() - starttime)
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378 accumulatornames = ('walltime (seconds)','total reads read','total reads counted','number of contigs','total empty reads','total ambiguous reads','total low quality reads',
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379 'total not aligned reads','total not unique mapping reads','extra filtered reads','empty contigs')
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380 accums = (walltime,nreads,totalc,len(contigs),empty,ambiguous,lowqual,notaligned,nonunique,filtered,emptycontigs)
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381 fracs = (1.0,1.0,float(totalc)/nreads,1.0,float(empty)/nreads,float(ambiguous)/nreads,float(lowqual)/nreads,float(notaligned)/nreads,float(nonunique)/nreads,float(filtered)/nreads,float(emptycontigs)/len(contigs))
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382 notes = ['%s = %d (%2.3f)' % (accumulatornames[i],x,100.0*fracs[i]) for i,x in enumerate(accums)]
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383 print >> sys.stdout, '\n'.join(notes)
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384 sys.exit(0)
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