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7
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1 '''
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2 the scripts takes two files as input, and compute the coverage of
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3 features in input 1 across features in input 2. Features in input 2 are
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4 divided into bins and coverage is computed for each bin.
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5
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6 please check the help information by typing:
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7
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8 python align.py -h
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9
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10
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11 requirement:
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12 please install the following tools first:
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13 bedtools: for read/region overlapping, http://code.google.com/p/bedtools/
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14
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15 '''
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16
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17 import os,sys,os.path
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18 from optparse import OptionParser
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19
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20 def lineCount(filename):
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21 with open(filename) as f:
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22 for i, l in enumerate(f):
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23 pass
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24 return i + 1
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25
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26 def combineFilename(f1,f2):
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27 '''
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28 fuse two file names into one
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29 '''
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30 return f1.split('/')[-1]+'-'+f2.split('/')[-1]
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31
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32 def checkFormat(filename1,filename2,input1format):
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33 '''
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34 check the format of input files
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35 '''
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36
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37 # file1
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38 # read the first line, see how many filds
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39 ncol1 = 6
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40 if input1format == "BED":
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41 f = open(filename1)
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42 line = f.readline().strip().split('\t')
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43 ncol1 = len(line)
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44 if ncol1 < 3:
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45 print "ERROR: "+filename1+" has only "+str(ncol1)+" columns (>=3 required). Make sure it has NO header line and is TAB-delimited."
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46 sys.exit(1)
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47 f.close()
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48
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49 # file2
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50 f = open(filename2)
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51 line = f.readline().strip().split('\t')
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52 ncol2 = len(line)
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53 if ncol2 < 3:
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54 print "ERROR: "+filename2+" has only "+str(ncol2)+" columns (>=3 required). Make sure it has NO header line and is TAB-delimited."
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55 sys.exit(1)
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56
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57 return ncol1,ncol2
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58
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59
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60 def makeBed(filename,ncol):
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61 '''
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62 add up to 6 column
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63 '''
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64 f = open(filename)
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65 outfile = filename+'.tmp.bed'
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66 outf = open(outfile,'w')
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67 if ncol == 3:
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68 for line in f:
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69 outf.write(line.strip()+'\t.\t0\t+\n')
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70 elif ncol == 4:
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71 for line in f:
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72 outf.write(line.strip()+'\t0\t+\n')
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73 if ncol == 5:
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74 for line in f:
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75 outf.write(line.strip()+'\t+\n')
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76 f.close()
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77 outf.close()
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78 return outfile
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79
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80 def makeWindow(filename,window):
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81
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82 outfile = filename+'-window='+str(window)+'.tmp.bed'
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83 if not os.path.exists(outfile):
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84 f=open(filename)
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85 out = open(outfile,'w')
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86 lines = f.readlines()
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87 if 'track' in lines[0]:
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88 del lines[0]
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89 for line in lines:
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90 flds = line.strip().split('\t')
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91
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92 #new position
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93 center = (int(flds[1]) + int(flds[2]))/2
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94 start = center - window
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95 end = center + window
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96 if start >= 0:
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97 flds[1] = str(start)
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98 flds[2] = str(end)
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99 out.write('\t'.join(flds)+'\n')
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100 f.close()
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101 out.close()
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102 return outfile
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103
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104 def groupReadsMapped2aRegion(filename,ncol):
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105 '''
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106 read output from intersectBED
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107 find all reads mapped to each region
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108 '''
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109 try:
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110 f=open(filename)
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111 #If filename cannot be opened, print an error message and exit
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112 except IOError:
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113 print "could not open",filename,"Are you sure this file exists?"
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114 sys.exit(1)
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115 lines = f.readlines()
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116
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117 allReadsStart = {}
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118 allReadsEnd = {}
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119 regionStrand = {}
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120 regionStart = {}
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121 regionEnd = {}
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122
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123 for line in lines:
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124 flds = line.strip().split('\t')
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125 key = '_'.join(flds[ncol:(ncol+4)])
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126 if not allReadsStart.has_key(key):
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127 allReadsStart[key] = list()
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128 allReadsEnd[key] = list()
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129 #print flds[ncol+0],flds[ncol+1],flds[ncol+2]
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130 allReadsStart[key].append(int(flds[1]))
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131 allReadsEnd[key].append(int(flds[2]))
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132 regionStrand[key] = flds[ncol+5]
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133 regionStart[key] = int(flds[ncol+1])
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134 regionEnd[key] = int(flds[ncol+2])
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135 return (allReadsStart,allReadsEnd,regionStrand,regionStart,regionEnd)
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136
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137
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138 def createRegionProfile(allReadsStart,allReadsEnd,regionStrand,regionStart,regionEnd,nbins):
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139 '''
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140 each region is divided into nbins
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141 compute the number of reads covering each bin for each region
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142 '''
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143 RegionProfile = {}
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144 nRead = {} # num of all reads in the region
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145 for region in allReadsStart.keys():
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146 RegionProfile[region] = [0]*nbins
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147 nRead[region] = len(allReadsStart[region])
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148 #print region,nRead[region],allReadsStart[region]
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149 for i in range(nRead[region]):
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150 RegionProfile[region] = updateRegionCount(RegionProfile[region],allReadsStart[region][i],allReadsEnd[region][i],regionStart[region],regionEnd[region],regionStrand[region],nbins)
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151 return RegionProfile,nRead
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152
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153 def updateRegionCount(RegionCount,readStart,readEnd,regionStart,regionEnd,strand,nbins):
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154 '''
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155 each region is divided into nbins,
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156 add 1 to each bin covered by the read
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157 '''
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158 L = regionEnd-regionStart
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159 start = int(nbins*(readStart-regionStart)/L)
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160 end = int(nbins*(readEnd-regionStart)/L)
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161 if start < 0:
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162 start = 0
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163 if end > nbins:
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164 end = nbins
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165 if strand == '-':
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166 for i in range(start,end):
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167 RegionCount[nbins-1-i] = RegionCount[nbins-1-i] + 1
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168 else: # if the 6th column of the input is not strand, will treat as + strand by default
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169 for i in range(start,end):
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170 RegionCount[i] = RegionCount[i] + 1
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171 return RegionCount
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172
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173 def saveProfile(filename,Profile,nRegion):
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174 out = open(filename,'w')
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175 for regionType in Profile.keys():
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176 #print Profile[regionType]
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177 out.write(regionType+'\t'+str(nRegion[regionType])+'\t'+'\t'.join(map(str,Profile[regionType]))+'\n')
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178
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179 def saveSummary(filename,Profile,nbin):
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180 out = open(filename+'.summary','w')
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181
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182 nfeat = len(Profile)
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183 summaryprofile = [0]*nbin
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184 for regionType in Profile.keys():
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185 for i in range(nbin):
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186 summaryprofile[i] += Profile[regionType][i]
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187 out.write(filename+'\t'+str(nfeat)+'\t'+'\t'.join(map(str,summaryprofile))+'\n')
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188 out.close()
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189 # calculate standard error
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190 out = open(filename+'.standarderror','w')
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191 sd = [0.0]*nbin
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192 u = [0.0]*nbin
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193 for i in range(nbin):
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194 u[i] = float(summaryprofile[i])/nfeat
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195 for regionType in Profile.keys():
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196 sd[i] = sd[i] + (Profile[regionType][i] - u[i])**2
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197 sd[i] = sd[i]**0.5 / nfeat
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198 out.write(filename+'\t'+str(nfeat)+'\t'+'\t'.join(map(str,sd))+'\n')
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199 out.close()
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200
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201 def main():
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202 usage = "usage: %prog [options] -a inputA -b inputB"
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203 parser = OptionParser(usage)
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204 parser.add_option("-a", dest="inputA",
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205 help="(required) input file A, interval (first 3 columns are chrN, start and end) or BAM format. The script computes the depth of coverage of features in file A across the features in file B" )
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206 parser.add_option("-b",dest="inputB",
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207 help="(required) input file B, interval file" )
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208 parser.add_option("-f",dest="aformat",default="BED",
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209 help="Format of input file A. Can be BED (default) or BAM")
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210 parser.add_option("-w",type='int',dest="window",
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211 help="Generate new inputB by making a window of 2 x WINDOW bp (in total) flanking the center of each input feature" )
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212 parser.add_option("-n", type="int", dest="nbins",default=100,
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213 help="number of bins. Features in B are binned, and the coverage is computed for each bin. Default is 100")
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214 parser.add_option("-s",action="store_true", dest="strandness",
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215 help="enforce strandness: require overlapping on the same strand. Default is off")
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216 parser.add_option("-p",action="store_true", dest="plot",default=False,
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217 help="load existed intersectBed outputfile")
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218 parser.add_option("-q", action="store_true", dest="quiet",default=False,
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219 help="suppress output on screen")
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220 parser.add_option("-o", dest="output_data",
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221 help="(optional) output coverage file (txt) name." )
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222 parser.add_option("-v", dest="output_plot",
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223 help="(optional) output plot (pdf) file name." )
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224 parser.add_option("-l", dest="plot_title", default="",
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225 help="(optional) output title of the plot." )
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226 parser.add_option("--ylim", dest="ylim", default="min,max",
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227 help="(optional) ylim of the plot" )
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228 parser.add_option("--summary-only", action="store_true", dest="summary_only",default=False,
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229 help="save profile summary only (no data for individual features)")
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230 parser.add_option("--compute-se", action="store_true", dest="compute_se",default=False,
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231 help="compute and plot standard deviation for each bin. used when --summary-only is on")
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232 parser.add_option("--profile-only", action="store_true", dest="profile_only",default=False,
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233 help="save profile only (no plot)")
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234 parser.add_option("--span", type="float", dest="span",default=0.1,
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235 help="loess span smooth parameter, 0.1 ~ 1")
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236
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237 (options, args) = parser.parse_args()
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238
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239 if options.inputA == None or options.inputB == None:
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240 parser.error("Please specify two input files!!")
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241
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242 if not options.quiet:
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243 print "checking input file format..."
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244
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245 ncol,ncol2 = checkFormat(options.inputA ,options.inputB,options.aformat)
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246
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247 if ncol2 < 6:
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248 options.inputB = makeBed(options.inputB,ncol2)
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249 if not options.quiet:
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250 print "fill up 6 columns"
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251
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252 if options.window > 0:
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253 if not options.quiet:
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254 print "making windows from "+options.inputB+"..."
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255 options.inputB = makeWindow(options.inputB,options.window)
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256
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257 output = combineFilename(str(options.inputA),str(options.inputB))
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258
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259 if not options.plot:
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260 if options.aformat == "BAM":
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261 cmd = "intersectBed -abam "+str(options.inputA)+" -b "+str(options.inputB) + ' -bed -split '
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262 else:
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263 cmd = "intersectBed -a "+str(options.inputA)+" -b "+str(options.inputB)
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264 if options.strandness:
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265 cmd = cmd + ' -s'
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266 cmd = cmd +" -wo > "+ output+'-intersect.tmp.bed'
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267 if not options.quiet:
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268 print "search for overlappings: "+cmd
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269 status = os.system(cmd)
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270 if status != 0:
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271 sys.exit(1)
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272
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273
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274 if not options.quiet:
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275 print 'group reads mapped to the same region...'
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276
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277 allReadsStart,allReadsEnd,regionStrand,regionStart,regionEnd = groupReadsMapped2aRegion(output+'-intersect.tmp.bed',ncol)
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278
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279 if len(allReadsStart) == 0:
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280 if not options.quiet:
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281 print 'no overlap found!!'
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282 os.system('rm *tmp.*')
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283 sys.exit(1)
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284
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285 if not options.quiet:
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286 print 'count number of reads mapped to each bin...'
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287
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288 RegionProfile,nRead = createRegionProfile(allReadsStart,allReadsEnd,regionStrand,regionStart,regionEnd,options.nbins)
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289
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290 if options.output_data == None:
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291 options.output_data = output+'.txt'
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292
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293 if options.summary_only:
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294 saveSummary(options.output_data,RegionProfile,options.nbins)
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295
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296 else:
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297 saveProfile(options.output_data,RegionProfile,nRead)
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298
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299 if not options.quiet:
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300 print 'results saved to: '+ options.output_data
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301
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302 if not (options.summary_only or options.profile_only ):
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303 # visualize
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304
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305 if options.window < 1:
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306 xlab = 'relative position (bins)'
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307 else:
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308 xlab = 'relative position (bp)'
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309
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310 if options.output_plot == None:
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311 options.output_plot = output+'.pdf'
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312
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313 title = options.plot_title+'\n n = '+str(len(RegionProfile))
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314
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315 rscript = open("tmp.r","w")
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316 rscript.write("x <- read.table('"+options.output_data+"')\n")
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317 rscript.write("pdf('"+options.output_plot+"')\n")
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318 rscript.write("avg <- colSums(x[,3:ncol(x)])/nrow(x)\n")
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319 rscript.write("err <- sd(x[,3:ncol(x)])/sqrt(nrow(x))\n")
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320
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321 if options.window == 0:
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322 rscript.write("xticks <- seq("+str(options.nbins)+")\n")
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323 else:
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324 rscript.write("xticks <- seq("+str(-options.window)+","+str(options.window)+",length.out="+str(options.nbins)+")\n")
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325
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326 if options.ylim != 'min,max':
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327 rscript.write("ylim=c("+options.ylim+")\n")
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328 else:
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329 rscript.write("ylim=c(min(avg-err),max(avg+err))\n")
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330 rscript.write("par(cex=1.5)\n")
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331 #smooth
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332 if options.span >= 0.1:
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333 rscript.write("avg = loess(avg~xticks,span="+str(options.span)+")$fitted\n")
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334 rscript.write("err = loess(err~xticks,span="+str(options.span)+")$fitted\n")
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335 rscript.write("plot(xticks,avg,ylab='average coverage',main='"+title+"',xlab='"+xlab+"',type='l',lwd=0,ylim=ylim)\n")
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336 rscript.write("polygon(c(xticks,rev(xticks)),c(avg+err,rev(avg-err)),col='slateblue1',border=NA)\n")
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337 rscript.write("lines(xticks,avg,type='l',lwd=1)\n")
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338 #rscript.write("xticks <- barplot(avg,names.arg=seq("+str(options.nbins)+"),ylab='average coverage',main='"+title+"',xlab='"+xlab+"',,ylim=c(min(avg-err),max(avg+err)))\n")
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339 #rscript.write("arrows(xticks,avg+err, xticks, avg-err, angle=90, code=3, length=0.0,col='green')\n")
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340 #rscript.write("lines(xticks,avg,lwd=2)\n")
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341 #rscript.write("lines(xticks,avg-err,col='green')\n")
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342 #rscript.write("lines(xticks,avg+err,col='green')\n")
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343 rscript.write("dev.off()\n")
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344 rscript.close()
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345
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346 os.system("R --vanilla < tmp.r")
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347
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348 # remove intermediate output
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349 os.system('rm *tmp.*')
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350
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351
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352 if __name__ == "__main__":
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353 main()
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