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1 #!/usr/bin/env python
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2 """
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3 #
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4 #------------------------------------------------------------------------------
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5 # University of Minnesota
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6 # Copyright 2017, Regents of the University of Minnesota
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7 #------------------------------------------------------------------------------
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8 # Author:
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9 #
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10 # James E Johnson
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11 #
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12 #------------------------------------------------------------------------------
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13 """
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14
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15 import argparse
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16 import sys
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17 from time import sleep
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18
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19 from Bio.Seq import translate
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20
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21 import requests
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22
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23 import digest
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24
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25
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26 server = "https://rest.ensembl.org"
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27 ext = "/info/assembly/homo_sapiens?"
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28 max_region = 5000000
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29
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30
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31 def ensembl_rest(ext, headers):
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32 if True: print >> sys.stderr, "%s" % ext
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33 r = requests.get(server+ext, headers=headers)
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34 if r.status_code == 429:
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35 print >> sys.stderr, "response headers: %s\n" % r.headers
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36 if 'Retry-After' in r.headers:
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37 sleep(r.headers['Retry-After'])
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38 r = requests.get(server+ext, headers=headers)
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39 if not r.ok:
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40 r.raise_for_status()
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41 return r
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42
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43
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44 def get_species():
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45 results = dict()
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46 ext = "/info/species"
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47 req_header = {"Content-Type": "application/json"}
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48 r = ensembl_rest(ext, req_header)
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49 for species in r.json()['species']:
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50 results[species['name']] = species
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51 print >> sys.stdout,\
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52 "%s\t%s\t%s\t%s\t%s"\
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53 % (species['name'], species['common_name'],
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54 species['display_name'],
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55 species['strain'],
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56 species['taxon_id'])
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57 return results
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58
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59
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60 def get_biotypes(species):
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61 biotypes = []
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62 ext = "/info/biotypes/%s?" % species
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63 req_header = {"Content-Type": "application/json"}
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64 r = ensembl_rest(ext, req_header)
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65 for entry in r.json():
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66 if 'biotype' in entry:
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67 biotypes.append(entry['biotype'])
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68 return biotypes
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69
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70
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71 def get_toplevel(species):
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72 coord_systems = dict()
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73 ext = "/info/assembly/%s?" % species
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74 req_header = {"Content-Type": "application/json"}
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75 r = ensembl_rest(ext, req_header)
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76 toplevel = r.json()
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77 for seq in toplevel['top_level_region']:
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78 if seq['coord_system'] not in coord_systems:
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79 coord_systems[seq['coord_system']] = dict()
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80 coord_system = coord_systems[seq['coord_system']]
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81 coord_system[seq['name']] = int(seq['length'])
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82 return coord_systems
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83
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84
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85 def get_transcripts_bed(species, refseq, start, length,params=None):
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86 bed = []
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87 param = params if params else ''
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88 req_header = {"Content-Type": "text/x-bed"}
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89 regions = range(start, length, max_region)
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90 if not regions or regions[-1] < length:
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91 regions.append(length)
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92 for end in regions[1:]:
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93 ext = "/overlap/region/%s/%s:%d-%d?feature=transcript;%s"\
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94 % (species, refseq, start, end, param)
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95 start = end + 1
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96 r = ensembl_rest(ext, req_header)
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97 if r.text:
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98 bed += r.text.splitlines()
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99 return bed
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100
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101
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102 def get_seq(id, seqtype,params=None):
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103 param = params if params else ''
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104 ext = "/sequence/id/%s?type=%s;%s" % (id, seqtype,param)
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105 req_header = {"Content-Type": "text/plain"}
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106 r = ensembl_rest(ext, req_header)
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107 return r.text
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108
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109
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110 def get_cdna(id,params=None):
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111 return get_seq(id, 'cdna',params=params)
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112
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113
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114 def get_cds(id,params=None):
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115 return get_seq(id, 'cds',params=params)
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116
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117
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118 def bed_from_line(line):
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119 fields = line.rstrip('\r\n').split('\t')
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120 (chrom, chromStart, chromEnd, name, score, strand,
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121 thickStart, thickEnd, itemRgb,
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122 blockCount, blockSizes, blockStarts) = fields[0:12]
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123 bed_entry = BedEntry(chrom=chrom, chromStart=chromStart, chromEnd=chromEnd,
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124 name=name, score=score, strand=strand,
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125 thickStart=thickStart, thickEnd=thickEnd,
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126 itemRgb=itemRgb,
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127 blockCount=blockCount,
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128 blockSizes=blockSizes.rstrip(','),
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129 blockStarts=blockStarts.rstrip(','))
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130 return bed_entry
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131
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132
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133 class BedEntry(object):
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134 def __init__(self, chrom=None, chromStart=None, chromEnd=None,
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135 name=None, score=None, strand=None,
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136 thickStart=None, thickEnd=None, itemRgb=None,
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137 blockCount=None, blockSizes=None, blockStarts=None):
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138 self.chrom = chrom
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139 self.chromStart = int(chromStart)
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140 self.chromEnd = int(chromEnd)
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141 self.name = name
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142 self.score = int(score) if score is not None else 0
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143 self.strand = '-' if str(strand).startswith('-') else '+'
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144 self.thickStart = int(thickStart) if thickStart else self.chromStart
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145 self.thickEnd = int(thickEnd) if thickEnd else self.chromEnd
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146 self.itemRgb = str(itemRgb) if itemRgb is not None else r'100,100,100'
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147 self.blockCount = int(blockCount)
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148 if isinstance(blockSizes, str) or isinstance(blockSizes, unicode):
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149 self.blockSizes = [int(x) for x in blockSizes.split(',')]
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150 elif isinstance(blockSizes, list):
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151 self.blockSizes = [int(x) for x in blockSizes]
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152 else:
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153 self.blockSizes = blockSizes
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154 if isinstance(blockStarts, str) or isinstance(blockSizes, unicode):
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155 self.blockStarts = [int(x) for x in blockStarts.split(',')]
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156 elif isinstance(blockStarts, list):
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157 self.blockStarts = [int(x) for x in blockStarts]
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158 else:
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159 self.blockStarts = blockStarts
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160 self.seq = None
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161 self.pep = None
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162
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163 def __str__(self):
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164 return '%s\t%d\t%d\t%s\t%d\t%s\t%d\t%d\t%s\t%d\t%s\t%s' % (
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165 self.chrom, self.chromStart, self.chromEnd,
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166 self.name, self.score, self.strand,
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167 self.thickStart, self.thickEnd, str(self.itemRgb), self.blockCount,
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168 ','.join([str(x) for x in self.blockSizes]),
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169 ','.join([str(x) for x in self.blockStarts]))
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170
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171 # (start, end)
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172 def get_subrange(self, tstart, tstop, debug=False):
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173 chromStart = self.chromStart
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174 chromEnd = self.chromEnd
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175 if debug:
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176 print >> sys.stderr, "%s" % (str(self))
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177 r = range(self.blockCount)
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178 if self.strand == '-':
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179 r.reverse()
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180 bStart = 0
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181 bEnd = 0
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182 for x in r:
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183 bEnd = bStart + self.blockSizes[x]
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184 if bStart <= tstart < bEnd:
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185 if self.strand == '+':
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186 chromStart = self.chromStart + self.blockStarts[x] +\
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187 (tstart - bStart)
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188 else:
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189 chromEnd = self.chromStart + self.blockStarts[x] +\
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190 self.blockSizes[x] - (tstart - bStart)
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191 if bStart <= tstop < bEnd:
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192 if self.strand == '+':
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193 chromEnd = self.chromStart + self.blockStarts[x] +\
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194 (tstop - bStart)
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195 else:
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196 chromStart = self.chromStart + self.blockStarts[x] +\
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197 self.blockSizes[x] - (tstop - bStart)
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198 if debug:
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199 print >> sys.stderr,\
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200 "%3d %s\t%d\t%d\t%d\t%d\t%d\t%d"\
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201 % (x, self.strand, bStart, bEnd,
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202 tstart, tstop, chromStart, chromEnd)
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203 bStart += self.blockSizes[x]
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204 return(chromStart, chromEnd)
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205
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206 # get the blocks for sub range
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207 def get_blocks(self, chromStart, chromEnd):
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208 tblockCount = 0
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209 tblockSizes = []
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210 tblockStarts = []
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211 for x in range(self.blockCount):
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212 bStart = self.chromStart + self.blockStarts[x]
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213 bEnd = bStart + self.blockSizes[x]
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214 if bStart > chromEnd:
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215 break
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216 if bEnd < chromStart:
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217 continue
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218 cStart = max(chromStart, bStart)
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219 tblockStarts.append(cStart - chromStart)
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220 tblockSizes.append(min(chromEnd, bEnd) - cStart)
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221 tblockCount += 1
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222 return (tblockCount, tblockSizes, tblockStarts)
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223
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224 def trim(self, tstart, tstop, debug=False):
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225 (tchromStart, tchromEnd) =\
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226 self.get_subrange(tstart, tstop, debug=debug)
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227 (tblockCount, tblockSizes, tblockStarts) =\
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228 self.get_blocks(tchromStart, tchromEnd)
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229 tbed = BedEntry(
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230 chrom=self.chrom, chromStart=tchromStart, chromEnd=tchromEnd,
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231 name=self.name, score=self.score, strand=self.strand,
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232 thickStart=tchromStart, thickEnd=tchromEnd, itemRgb=self.itemRgb,
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233 blockCount=tblockCount,
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234 blockSizes=tblockSizes, blockStarts=tblockStarts)
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235 if self.seq:
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236 ts = tchromStart-self.chromStart
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237 te = tchromEnd - tchromStart + ts
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238 tbed.seq = self.seq[ts:te]
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239 return tbed
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240
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241
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242 def __main__():
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243 parser = argparse.ArgumentParser(
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244 description='Retrieve Ensembl cDNAs and three frame translate')
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245 parser.add_argument(
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246 '-s', '--species', default='human',
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247 help='Ensembl Species to retrieve')
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248 parser.add_argument(
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249 '-B', '--biotypes', action='append', default=[],
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250 help='Restrict Ensembl biotypes to retrieve')
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251 parser.add_argument(
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252 '-i', '--input', default=None,
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253 help='Use BED instead of retrieving cDNA from ensembl (-) for stdin')
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254 parser.add_argument(
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255 '-t', '--transcripts', default=None,
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256 help='Path to output cDNA transcripts.bed (-) for stdout')
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257 parser.add_argument(
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258 '-r', '--raw', action='store_true',
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259 help='Report transcript exacty as returned from Ensembl')
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260 parser.add_argument(
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261 '-f', '--fasta', default=None,
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262 help='Path to output translations.fasta')
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263 parser.add_argument(
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264 '-b', '--bed', default=None,
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265 help='Path to output translations.bed')
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266 parser.add_argument(
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267 '-m', '--min_length', type=int, default=7,
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268 help='Minimum length of protein translation to report')
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269 parser.add_argument(
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270 '-e', '--enzyme', default=None,
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271 help='Digest translation with enzyme')
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272 parser.add_argument(
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273 '-a', '--all', action='store_true',
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274 help='Include reference protein translations')
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275 parser.add_argument('-v', '--verbose', action='store_true', help='Verbose')
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276 parser.add_argument('-d', '--debug', action='store_true', help='Debug')
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277 args = parser.parse_args()
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278 # print >> sys.stderr, "args: %s" % args
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279 species = args.species
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280 input_rdr = None
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281 if args.input is not None:
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282 input_rdr = open(args.input, 'r') if args.input != '-' else sys.stdin
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283 tx_wtr = None
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284 if args.transcripts is not None:
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285 tx_wtr = open(args.transcripts, 'w')\
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286 if args.transcripts != '-' else sys.stdout
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287 fa_wtr = open(args.fasta, 'w') if args.fasta is not None else None
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288 bed_wtr = open(args.bed, 'w') if args.bed is not None else None
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289
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290 enzyme = digest.expasy_rules.get(args.enzyme,args.enzyme)
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291
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292 # print >> sys.stderr, "args biotypes: %s" % args.biotypes
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293 biotypea = ['biotype=%s' % bt.strip() for biotype in args.biotypes for bt in biotype.split(',')]
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294 # print >> sys.stderr, "args biotypes: %s" % biotypea
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295 biotypes = ';'.join(['biotype=%s' % bt.strip() for biotype in args.biotypes for bt in biotype.split(',') if bt.strip()])
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296 # print >> sys.stderr, "biotypes: %s" % biotypes
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297
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298 translations = dict() # start : end : seq
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299
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300 def unique_prot(tbed, seq):
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301 if tbed.chromStart not in translations:
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302 translations[tbed.chromStart] = dict()
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303 translations[tbed.chromStart][tbed.chromEnd] = []
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304 translations[tbed.chromStart][tbed.chromEnd].append(seq)
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305 elif tbed.chromEnd not in translations[tbed.chromStart]:
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306 translations[tbed.chromStart][tbed.chromEnd] = []
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307 translations[tbed.chromStart][tbed.chromEnd].append(seq)
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308 elif seq not in translations[tbed.chromStart][tbed.chromEnd]:
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309 translations[tbed.chromStart][tbed.chromEnd].append(seq)
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310 else:
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311 return False
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312 return True
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313
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314 def translate_bed(bed):
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315 translate_count = 0
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316 if any([fa_wtr, bed_wtr]):
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317 transcript_id = bed.name
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318 refprot = None
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319 if not args.all:
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320 try:
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321 cds = get_cds(transcript_id)
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322 if len(cds) % 3 != 0:
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323 cds = cds[:-(len(cds) % 3)]
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324 refprot = translate(cds) if cds else None
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325 except:
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326 refprot = None
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327 cdna = get_cdna(transcript_id)
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328 cdna_len = len(cdna)
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329 for offset in range(3):
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330 seqend = cdna_len - (cdna_len - offset) % 3
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331 aaseq = translate(cdna[offset:seqend])
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332 aa_start = 0
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333 while aa_start < len(aaseq):
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334 aa_end = aaseq.find('*', aa_start)
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335 if aa_end < 0:
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336 aa_end = len(aaseq)
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337 prot = aaseq[aa_start:aa_end]
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338 if enzyme and refprot:
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339 frags = digest._cleave(prot,enzyme)
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340 for frag in reversed(frags):
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341 if frag in refprot:
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342 prot = prot[:prot.rfind(frag)]
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343 else:
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344 break
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345 if len(prot) < args.min_length:
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346 pass
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347 elif refprot and prot in refprot:
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348 pass
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349 else:
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350 tstart = aa_start*3+offset
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351 tend = aa_end*3+offset
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352 prot_acc = "%s_%d_%d" % (transcript_id, tstart, tend)
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353 tbed = bed.trim(tstart, tend)
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354 if args.all or unique_prot(tbed, prot):
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355 translate_count += 1
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356 tbed.name = prot_acc
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357 bed_wtr.write("%s\t%s\n" % (str(tbed), prot))
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358 bed_wtr.flush()
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359 fa_id = ">%s\n" % (prot_acc)
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360 fa_wtr.write(fa_id)
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361 fa_wtr.write(prot)
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362 fa_wtr.write("\n")
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363 fa_wtr.flush()
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364 aa_start = aa_end + 1
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365 return translate_count
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366
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367 if input_rdr:
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368 translation_count = 0
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369 for i, bedline in enumerate(input_rdr):
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370 try:
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371 bed = bed_from_line(bedline)
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372 translation_count += translate_bed(bed)
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373 except:
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374 print >> sys.stderr, "BED format error: %s\n" % bedline
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375 if args.debug or (args.verbose and any([fa_wtr, bed_wtr])):
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376 print >> sys.stderr,\
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377 "%s\tcDNA translations:%d" % (species, translation_count)
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378 else:
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379 coord_systems = get_toplevel(species)
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380 if 'chromosome' in coord_systems:
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381 for ref in sorted(coord_systems['chromosome'].keys()):
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382 length = coord_systems['chromosome'][ref]
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383 if not any([tx_wtr, fa_wtr, bed_wtr]):
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384 print >> sys.stderr,\
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385 "%s\t%s\tlength: %d" % (species, ref, length)
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386 continue
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387 if args.debug:
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388 print >> sys.stderr,\
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389 "Retrieving transcripts: %s\t%s\tlength: %d"\
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390 % (species, ref, length)
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391 translation_count = 0
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392 start = 0
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393 regions = range(start, length, max_region)
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|
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394 if not regions or regions[-1] < length:
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|
395 regions.append(length)
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|
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396 for end in regions[1:]:
|
|
|
397 bedlines = get_transcripts_bed(species, ref, start, end, params=biotypes)
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|
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398 if args.verbose or args.debug:
|
|
|
399 print >> sys.stderr,\
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|
|
400 "%s\t%s\tstart: %d\tend: %d\tcDNA transcripts:%d"\
|
|
|
401 % (species, ref, start, end, len(bedlines))
|
|
|
402 # start, end, seq
|
|
|
403 for i, bedline in enumerate(bedlines):
|
|
|
404 try:
|
|
|
405 bed = bed_from_line(bedline)\
|
|
|
406 if any([not args.raw, fa_wtr, bed_wtr])\
|
|
|
407 else None
|
|
|
408 if tx_wtr:
|
|
|
409 tx_wtr.write(bedline if args.raw else str(bed))
|
|
|
410 tx_wtr.write("\n")
|
|
|
411 tx_wtr.flush()
|
|
|
412 if bed:
|
|
|
413 translation_count += translate_bed(bed)
|
|
|
414 except Exception as e:
|
|
|
415 print >> sys.stderr,\
|
|
|
416 "BED error (%s) : %s\n" % (e, bedline)
|
|
|
417 start = end + 1
|
|
|
418
|
|
|
419 if args.debug or (args.verbose and any([fa_wtr, bed_wtr])):
|
|
|
420 print >> sys.stderr,\
|
|
|
421 "%s\t%s\tlength: %d\tcDNA translations:%d"\
|
|
|
422 % (species, ref, length, translation_count)
|
|
|
423
|
|
|
424
|
|
|
425 if __name__ == "__main__":
|
|
|
426 __main__()
|
