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1 # Python3 script which takes in an annotation file(gtf/gff3) and a transcriptomic fasta file
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2 # and produces an sqlite file which can be uploaded to Trips-Viz
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3 # All co-ordinates produced are 1 based
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4 # All start codon positions (including cds_start) should be at the first nucleotide of the codon
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5 # All stop codon positions (including cds_stop) should be at the last nucleotide of the codon
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6 import sys
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7 import re
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8 import sqlite3
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9 from intervaltree import Interval, IntervalTree
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10 import itertools
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11 from sqlitedict import SqliteDict
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12 import os
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13
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14 organism = sys.argv[1]
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15 # This should be a GTF or GFF3 file
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16 annotation_file = open(sys.argv[2], "r")
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17 # This needs to be the transcriptomic fasta file
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18 fasta_file = open(sys.argv[3], "r")
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19 # This value will be added used to create UTRs of this length, useful when looking at transcriptomes without annotated UTRs
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20 pseudo_utr_len = int(sys.argv[4])
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21 # An example of a transcript_id from the annotation file, e.g ENST000000123456
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22 user_transcript_id = sys.argv[5]
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23 # An example of a gene name from the annotation file
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24 user_gene_name = sys.argv[6]
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25 # Set to true if transcript version is included in transcript_id, e.g: ENST000000123456.1
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26 TRAN_VERSION = True
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27
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28
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29 if os.path.isfile("{}.sqlite".format(organism)):
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30 print("{}.sqlite already exists".format(organism))
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31 sys.exit()
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32
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33
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34 # old_exons = SqliteDict(
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35 # "/home/DATA/www/tripsviz/tripsviz/trips_annotations/mus_musculus/transcriptomic_to_genomic.sqlite"
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36 # )
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37
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38
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39 delimiters = {
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40 "transcripts": {"before": [], "after": ["."], "annot_types": ["cds", "utr"]},
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41 "genes": {"before": [], "after": ['"'], "annot_types": ["lnc_rna"]},
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42 }
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43
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44 punctuation = [";", " ", "-", ":", "-", ".", "=", "\t"]
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45 # Find delimiters in the annotation and fasta files using the user_transcript_id
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46 # and user_gene_name examples given by user.
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47 for line in annotation_file:
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48 if user_transcript_id in line:
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49 tabsplitline = line.split("\t")
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50 annot_type = tabsplitline[2]
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51 if annot_type not in delimiters["transcripts"]["annot_types"]:
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52 delimiters["transcripts"]["annot_types"].append(annot_type.lower())
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53 splitline = line.split(user_transcript_id)
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54 before_delimiter = splitline[0]
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55 for item in punctuation:
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56 if item in before_delimiter:
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57 if len(before_delimiter.split(item)[-1]) >= 5:
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58 before_delimiter = before_delimiter.split(item)[-1]
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59 after_delimiter = splitline[1][:2]
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60 if (
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61 before_delimiter not in delimiters["transcripts"]["before"]
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62 and len(before_delimiter) >= 5
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63 ):
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64 delimiters["transcripts"]["before"].append(before_delimiter)
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65 if after_delimiter not in delimiters["transcripts"]["after"]:
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66 delimiters["transcripts"]["after"].append(after_delimiter)
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67 if user_gene_name in line:
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68 tabsplitline = line.split("\t")
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69 annot_type = tabsplitline[2]
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70 print("ANNOT TYPE", annot_type)
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71 if annot_type not in delimiters["genes"]["annot_types"]:
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72 delimiters["genes"]["annot_types"].append(annot_type.lower())
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73 splitline = line.split(user_gene_name)
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74 before_delimiter = splitline[0]
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75 for item in punctuation:
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76 if item in before_delimiter:
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77 if len(before_delimiter.split(item)[-1]) >= 5:
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78 before_delimiter = before_delimiter.split(item)[-1]
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79 after_delimiter = splitline[1][0]
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80 if (
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81 before_delimiter not in delimiters["genes"]["before"]
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82 and len(before_delimiter) >= 5
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83 ):
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84 delimiters["genes"]["before"].append(before_delimiter)
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85 if after_delimiter not in delimiters["genes"]["after"]:
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86 if after_delimiter in punctuation:
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87 delimiters["genes"]["after"].append(after_delimiter)
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88
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89 print("delimeters[genes]", delimiters["transcripts"]["annot_types"])
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90
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91 for line in fasta_file:
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92 if user_transcript_id in line:
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93 splitline = line.split(user_transcript_id)
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94 before_delimiter = splitline[0]
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95 for item in punctuation:
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96 if item in before_delimiter:
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97 if len(before_delimiter.split(item)[1]) >= 5:
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98 before_delimiter = before_delimiter.split(item)[1]
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99 after_delimiter = splitline[1][0]
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100 if (
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101 before_delimiter not in delimiters["transcripts"]["before"]
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102 and len(before_delimiter) >= 5
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103 ):
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104 delimiters["transcripts"]["before"].append(before_delimiter)
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105 if after_delimiter not in delimiters["transcripts"]["after"]:
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106 delimiters["transcripts"]["after"].append(after_delimiter)
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107 fasta_file.close()
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108 annotation_file.close()
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109
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110
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111 if delimiters["transcripts"]["before"] == []:
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112 print(
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113 "ERROR: No transcript_id with the name {} could be found in the annotation file".format(
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114 user_transcript_id
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115 )
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116 )
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117 sys.exit()
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118 if delimiters["genes"]["before"] == []:
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119 print(
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120 "ERROR: No gene with the name {} could be found in the annotation file".format(
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121 user_gene_name
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122 )
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123 )
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124 sys.exit()
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125
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126 master_dict = {}
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127 coding_dict = {}
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128 notinfasta = open("notinfasta.csv", "w")
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129
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130 # Given a nucleotide sequence returns the positions of all start and stop codons.
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131 def get_start_stops(transcript_sequence):
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132 transcript_sequence = transcript_sequence.upper()
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133 start_codons = ["ATG"]
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134 stop_codons = ["TAA", "TAG", "TGA"]
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135 seq_frames = {"starts": [], "stops": []}
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136 for codons, positions in ((start_codons, "starts"), (stop_codons, "stops")):
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137 if len(codons) > 1:
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138 pat = re.compile("|".join(codons))
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139 else:
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140 pat = re.compile(codons[0])
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141 for m in re.finditer(pat, transcript_sequence):
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142 # Increment position by 1, Frame 1 starts at position 1 not 0,
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143 # if it's a stop codon add another 2 so it points to the last nuc of the codon
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144 if positions == "starts":
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145 start = m.start() + 1
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146 else:
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147 start = m.start() + 3
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148 seq_frames[positions].append(start)
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149 return seq_frames
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150
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151
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152 # parse fasta to get the nucleotide sequence of transcripts and the positions of start/stop codons.
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153 fasta_file = open(sys.argv[3], "r")
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154 read_fasta = fasta_file.read()
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155 split_fasta = read_fasta.split(">")
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156 for entry in split_fasta[1:]:
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157 newline_split = entry.split("\n")
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158 tran = newline_split[0]
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159 for item in delimiters["transcripts"]["after"]:
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160 if item in tran:
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161 tran = tran.split(item)[0]
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162 tran = tran.replace("-", "_").replace("(", "").replace(")", "")
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163 seq = "".join(newline_split[1:])
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164 if "_PAR_Y" in tran:
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165 tran += "_chrY"
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166 elif "_PAR_X" in tran:
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167 tran += "_chrX"
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168 tran = tran.upper()
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169 starts_stops = get_start_stops(seq)
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170 print("tran", tran)
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171 if tran not in master_dict:
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172 master_dict[tran] = {
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173 "utr": [],
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174 "cds": [],
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175 "exon": [],
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176 "start_codon": [],
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177 "stop_codon": [],
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178 "start_list": starts_stops["starts"],
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179 "stop_list": starts_stops["stops"],
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180 "transcript": [],
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181 "strand": "",
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182 "gene_name": "",
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183 "chrom": "",
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184 "seq": seq,
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185 "cds_start": "NULL",
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186 "cds_stop": "NULL",
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187 "length": len(seq),
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188 "principal": 0,
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189 "version": "NULL",
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190 }
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191
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192
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193 def to_ranges(iterable):
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194 tup_list = []
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195 iterable = sorted(set(iterable))
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196 for key, group in itertools.groupby(enumerate(iterable), lambda t: t[1] - t[0]):
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197 group = list(group)
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198 tup_list.append((group[0][1], group[-1][1]))
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199 return tup_list
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200
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201
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202 # parse annotation file to get chromsome, exon location and CDS info for each transcript
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203 def parse_gtf_file(annotation_file):
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204 for line in annotation_file:
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205 if line == "\n":
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206 continue
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207 if line[0] != "#":
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208 splitline = (line.replace("\n", "")).split("\t")
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209 chrom = splitline[0]
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210 try:
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211 annot_type = splitline[2].lower()
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212 except:
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213 print(
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214 "ERROR tried to index to second item in splitline: ",
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215 splitline,
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216 line,
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217 )
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218 sys.exit()
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219 # if annot_type not in ["cds", "utr", "exon", "transcript","five_prime_utr", "three_prime_utr","stop_codon","start_codon"]:
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220 # continue
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221 if (
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222 annot_type not in delimiters["transcripts"]["annot_types"]
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223 and annot_type not in delimiters["genes"]["annot_types"]
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224 ):
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225 continue
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226 if annot_type == "five_prime_utr" or annot_type == "three_prime_utr":
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227 annot_type = "utr"
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228 strand = splitline[6]
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229 if strand == "+":
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230 start = int(splitline[3])
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231 end = int(splitline[4])
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232 else:
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233 start = int(splitline[3]) + 1
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234 end = int(splitline[4]) + 1
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235 desc = splitline[8]
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236 tran = desc
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237 gene = desc
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238 for item in delimiters["transcripts"]["before"]:
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239 if item in tran:
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240 tran = tran.split(item)[1]
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241 for item in delimiters["transcripts"]["after"]:
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242 if item in tran:
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243 tran = tran.split(item)[0]
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244 if "." in tran and TRAN_VERSION == True:
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245 # print ("raw tran",tran)
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246 tran = tran.split(".")
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247 version = int(tran[-1].split("_")[0])
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248 tran = tran[0]
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249 else:
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250 version = "NULL"
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251 tran = tran.replace("-", "_").replace(".", "_")
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252 tran = tran.replace("(", "").replace(")", "")
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253 tran = tran.replace(" ", "").replace("\t", "")
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254 tran = tran.upper()
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255 tran = tran.replace("GENE_", "").replace("ID_", "")
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256 if "_PAR_Y" in desc:
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257 # print ("adding _PAR_Y to tran")
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258 tran = tran + "_PAR_Y"
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259 # print ("New tran ", tran)
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260 # if "PAR_Y" in line:
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261 # print (line)
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262 # #sys.exit()
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263 # print ("tran",tran,version)
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264 # if tran == "ENST00000316448":
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265 # print ("annot type",annot_type)
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266 # print ("appending exon to tran", start, end,line)
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267
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268 gene_found = False
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269
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270 if annot_type in delimiters["genes"]["annot_types"]:
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271 for item in delimiters["genes"]["before"]:
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272 if item in gene:
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273 gene_found = True
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274 gene = gene.split(item)[1]
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275 for item in delimiters["genes"]["after"]:
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276 if item in gene:
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277 gene = gene.split(item)[0]
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278 gene = gene.replace("'", "''")
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279 gene = gene.replace("GENE_", "")
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280 gene = gene.replace("ID_", "")
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281 gene = gene.upper()
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282 if tran in master_dict:
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283 master_dict[tran]["strand"] = strand
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284 if strand == "+":
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285 if annot_type in master_dict[tran]:
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286 master_dict[tran][annot_type].append((start, end))
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287 else:
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288 if annot_type in master_dict[tran]:
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289 master_dict[tran][annot_type].append((start, end))
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290 master_dict[tran]["chrom"] = chrom
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291 master_dict[tran]["version"] = version
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292 if gene_found == True:
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293 master_dict[tran]["gene_name"] = gene
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294 else:
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295 notinfasta.write("{}\n".format(tran))
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296
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297
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298 annotation_file = open(sys.argv[2], "r")
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299 parse_gtf_file(annotation_file)
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300
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301
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302 # remove transcripts that were in fasta file but not in annotation_file
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303 notinannotation = []
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304 for tran in master_dict:
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305 if master_dict[tran]["chrom"] == "":
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306 # print ("tran {} has no chrom :(".format(tran))
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307 notinannotation.append(tran)
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308 for tran in notinannotation:
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309 print(tran)
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310 del master_dict[tran]
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311 # Dictionary to store the coding status of a gene, if any transcript of this gene is coding, the value will be True
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312 coding_genes_dict = {}
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313 # parse master_dict to calculate length, cds_start/stop and exon junction positions
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314 for tran in master_dict:
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315 try:
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316 transeq = master_dict[tran]["seq"]
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317 except Exception as e:
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318 print("not in fasta", tran)
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319 notinfasta.write("{}\n".format(tran))
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320 continue
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321 exon_junctions = []
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322 total_length = len(transeq)
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323 three_len = 1
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324 five_len = 1
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325 strand = master_dict[tran]["strand"]
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326 if master_dict[tran]["gene_name"] == "":
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327 master_dict[tran]["gene_name"] = tran
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328 gene = master_dict[tran]["gene_name"]
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329 if gene not in coding_genes_dict:
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330 coding_genes_dict[gene] = False
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331
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332 if master_dict[tran]["cds"] == []:
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333 tran_type = "noncoding"
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334 cds_start = "NULL"
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335 cds_stop = "NULL"
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336 else:
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337 # get utr lengths from annotation
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338 tran_type = "coding"
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339 coding_genes_dict[gene] = True
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340 sorted_exons = sorted(master_dict[tran]["exon"])
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341 sorted_cds = sorted(master_dict[tran]["cds"])
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342
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343 min_cds = sorted_cds[0][0]
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344 # Some annotation files do not have utr annotation types, so fix that here if thats the case
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345 if master_dict[tran]["utr"] == []:
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346 for exon_tup in master_dict[tran]["exon"]:
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347 if exon_tup not in master_dict[tran]["cds"]:
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348 # Now check if this overlaps with any of the CDS exons
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349 overlap = False
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350 for cds_tup in master_dict[tran]["cds"]:
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351 if exon_tup[0] == cds_tup[0] and exon_tup[1] != cds_tup[1]:
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352 master_dict[tran]["utr"].append((cds_tup[1], exon_tup[1]))
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353 overlap = True
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354 if exon_tup[0] != cds_tup[0] and exon_tup[1] == cds_tup[1]:
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355 master_dict[tran]["utr"].append((exon_tup[0], cds_tup[0]))
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356 overlap = True
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357 if overlap == False:
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358 master_dict[tran]["utr"].append(exon_tup)
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359
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360 for tup in sorted(master_dict[tran]["utr"]):
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361 if tup[0] < min_cds:
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362 five_len += (tup[1] - tup[0]) + 1
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363 elif tup[0] > min_cds:
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364 three_len += (tup[1] - tup[0]) + 1
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365 else:
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366 pass
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367 if strand == "+":
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368 if len(sorted_exons) > 1:
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369 sorted_exons[0] = (
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370 sorted_exons[0][0] - pseudo_utr_len,
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371 sorted_exons[0][1],
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372 )
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373 sorted_exons[-1] = (
|
|
|
374 sorted_exons[-1][0],
|
|
|
375 sorted_exons[-1][1] + pseudo_utr_len,
|
|
|
376 )
|
|
|
377 else:
|
|
|
378 sorted_exons[0] = (
|
|
|
379 sorted_exons[0][0] - pseudo_utr_len,
|
|
|
380 sorted_exons[0][1] + pseudo_utr_len,
|
|
|
381 )
|
|
|
382 master_dict[tran]["exon"] = sorted_exons
|
|
|
383 cds_start = five_len + pseudo_utr_len
|
|
|
384 cds_stop = ((total_length - three_len) - pseudo_utr_len) + 4
|
|
|
385 elif strand == "-":
|
|
|
386 if len(sorted_exons) > 1:
|
|
|
387 sorted_exons[0] = (
|
|
|
388 (sorted_exons[0][0] - pseudo_utr_len),
|
|
|
389 sorted_exons[0][1],
|
|
|
390 )
|
|
|
391 sorted_exons[-1] = (
|
|
|
392 sorted_exons[-1][0],
|
|
|
393 (sorted_exons[-1][1] + pseudo_utr_len),
|
|
|
394 )
|
|
|
395 else:
|
|
|
396 sorted_exons[0] = (
|
|
|
397 (sorted_exons[0][0] - pseudo_utr_len),
|
|
|
398 (sorted_exons[0][1] + pseudo_utr_len),
|
|
|
399 )
|
|
|
400 master_dict[tran]["exon"] = sorted_exons
|
|
|
401 cds_start = three_len + pseudo_utr_len
|
|
|
402 cds_stop = ((total_length - (five_len)) - pseudo_utr_len) + 4
|
|
|
403 # if tran == "ENST00000381401":
|
|
|
404 # print ("cds start, cds stop, five_len, three_len",cds_start,cds_stop,five_len,three_len)
|
|
|
405 # #sys.exit()
|
|
|
406 else:
|
|
|
407 print("strand is unknown: {}".format(strand))
|
|
|
408 sys.exit()
|
|
|
409 # get exon junctions, cds is easy just get end of each tuple except last, same for utr except for if same as cds start/stop
|
|
|
410 total_intronic = 0
|
|
|
411 try:
|
|
|
412 if strand == "+":
|
|
|
413 tx_start = min(sorted(master_dict[tran]["exon"]))[0]
|
|
|
414 prev_end = tx_start
|
|
|
415 for tup in sorted(master_dict[tran]["exon"])[:-1]:
|
|
|
416 total_intronic += tup[0] - prev_end
|
|
|
417 exon_junctions.append(((tup[1]) - tx_start) - total_intronic)
|
|
|
418 prev_end = tup[1]
|
|
|
419 elif strand == "-":
|
|
|
420 tx_start = max(sorted(master_dict[tran]["exon"]))[-1]
|
|
|
421 prev_end = tx_start
|
|
|
422 for tup in (sorted(master_dict[tran]["exon"])[1:])[::-1]:
|
|
|
423 total_intronic += (tup[0] + 1) - prev_end
|
|
|
424 exon_junctions.append(((tup[1]) - tx_start) - total_intronic)
|
|
|
425 prev_end = tup[1]
|
|
|
426 except:
|
|
|
427 if strand == "+":
|
|
|
428 tx_start = min(sorted(master_dict[tran]["cds"]))[0]
|
|
|
429 prev_end = tx_start
|
|
|
430 for tup in sorted(master_dict[tran]["cds"])[:-1]:
|
|
|
431 total_intronic += tup[0] - prev_end
|
|
|
432 exon_junctions.append(((tup[1]) - tx_start) - total_intronic)
|
|
|
433 prev_end = tup[1]
|
|
|
434 elif strand == "-":
|
|
|
435 tx_start = max(sorted(master_dict[tran]["cds"]))[-1]
|
|
|
436 prev_end = tx_start
|
|
|
437 for tup in (sorted(master_dict[tran]["cds"])[1:])[::-1]:
|
|
|
438 total_intronic += (tup[0] + 1) - prev_end
|
|
|
439 exon_junctions.append(((tup[1]) - tx_start) - total_intronic)
|
|
|
440 prev_end = tup[1]
|
|
|
441 # This can happen when a coding transcript doesn't have a properly annotated 3' trailer
|
|
|
442 if cds_stop != "NULL":
|
|
|
443 if cds_stop > total_length:
|
|
|
444 cds_stop = total_length
|
|
|
445 if strand == "+" and cds_start != "NULL":
|
|
|
446 master_dict[tran]["cds_start"] = cds_start
|
|
|
447 master_dict[tran]["cds_stop"] = cds_stop
|
|
|
448 elif strand == "-" and cds_start != "NULL":
|
|
|
449 master_dict[tran]["cds_start"] = cds_start
|
|
|
450 master_dict[tran]["cds_stop"] = cds_stop
|
|
|
451
|
|
|
452 master_dict[tran]["strand"] = strand
|
|
|
453 master_dict[tran]["tran_type"] = tran_type
|
|
|
454 master_dict[tran]["exon_junctions"] = exon_junctions
|
|
|
455
|
|
|
456 longest_tran_dict = {}
|
|
|
457 for tran in master_dict:
|
|
|
458 try:
|
|
|
459 gene = master_dict[tran]["gene_name"]
|
|
|
460 except:
|
|
|
461 continue
|
|
|
462 if coding_genes_dict[gene] == True:
|
|
|
463 if "cds_start" in master_dict[tran]:
|
|
|
464 if (
|
|
|
465 master_dict[tran]["cds_stop"] != "NULL"
|
|
|
466 and master_dict[tran]["cds_start"] != "NULL"
|
|
|
467 ):
|
|
|
468 cds_length = (
|
|
|
469 master_dict[tran]["cds_stop"] - master_dict[tran]["cds_start"]
|
|
|
470 )
|
|
|
471 if gene not in longest_tran_dict:
|
|
|
472 longest_tran_dict[gene] = {"tran": tran, "length": cds_length}
|
|
|
473 else:
|
|
|
474 if cds_length > longest_tran_dict[gene]["length"]:
|
|
|
475 longest_tran_dict[gene] = {"tran": tran, "length": cds_length}
|
|
|
476 if cds_length == longest_tran_dict[gene]["length"]:
|
|
|
477 if (
|
|
|
478 master_dict[tran]["length"]
|
|
|
479 > master_dict[longest_tran_dict[gene]["tran"]]["length"]
|
|
|
480 ):
|
|
|
481 longest_tran_dict[gene] = {
|
|
|
482 "tran": tran,
|
|
|
483 "length": cds_length,
|
|
|
484 }
|
|
|
485 else:
|
|
|
486 length = master_dict[tran]["length"]
|
|
|
487 if gene not in longest_tran_dict:
|
|
|
488 longest_tran_dict[gene] = {"tran": tran, "length": length}
|
|
|
489 elif length > longest_tran_dict[gene]["length"]:
|
|
|
490 longest_tran_dict[gene] = {"tran": tran, "length": length}
|
|
|
491
|
|
|
492
|
|
|
493 for gene in longest_tran_dict:
|
|
|
494 longest_tran = longest_tran_dict[gene]["tran"]
|
|
|
495 master_dict[longest_tran]["principal"] = 1
|
|
|
496
|
|
|
497 gene_sample = []
|
|
|
498 for key in list(master_dict)[:10]:
|
|
|
499 try:
|
|
|
500 gene_sample.append(master_dict[key]["gene_name"])
|
|
|
501 except:
|
|
|
502 pass
|
|
|
503 print(master_dict)
|
|
|
504 print("Here is a sample of the transcript ids: {}".format(list(master_dict)[:10]))
|
|
|
505 print("Here is a sample of the gene names: {}".format(gene_sample))
|
|
|
506
|
|
|
507
|
|
|
508 # Takes a transcript, transcriptomic position and a master_dict (see ribopipe scripts) and returns the genomic position, positions should be passed 1 at a time.
|
|
|
509 def tran_to_genome(tran, start_pos, end_pos, master_dict):
|
|
|
510 pos_list = []
|
|
|
511 for i in range(start_pos, end_pos + 1):
|
|
|
512 pos_list.append(i)
|
|
|
513 genomic_pos_list = []
|
|
|
514 if tran in master_dict:
|
|
|
515 transcript_info = master_dict[tran]
|
|
|
516 else:
|
|
|
517 return ("Null", [])
|
|
|
518
|
|
|
519 chrom = transcript_info["chrom"]
|
|
|
520 strand = transcript_info["strand"]
|
|
|
521 exons = sorted(transcript_info["exon"])
|
|
|
522 # print ("chrom,strand,exons",chrom,strand,exons)
|
|
|
523 for pos in pos_list:
|
|
|
524 # print ("pos",pos)
|
|
|
525 if strand == "+":
|
|
|
526 exon_start = 0
|
|
|
527 for tup in exons:
|
|
|
528 # print ("tup",tup)
|
|
|
529 exon_start = tup[0]
|
|
|
530 exonlen = tup[1] - tup[0]
|
|
|
531 if pos > exonlen:
|
|
|
532 pos = (pos - exonlen) - 1
|
|
|
533 else:
|
|
|
534 break
|
|
|
535 # print ("appending exon_start-pos", exon_start, pos, exon_start+pos)
|
|
|
536 genomic_pos_list.append((exon_start + pos) - 1)
|
|
|
537 elif strand == "-":
|
|
|
538 exon_start = 0
|
|
|
539 for tup in exons[::-1]:
|
|
|
540 # print ("tup",tup)
|
|
|
541 exon_start = tup[1]
|
|
|
542 exonlen = tup[1] - tup[0]
|
|
|
543 # print ("exonlen",exonlen)
|
|
|
544 if pos > exonlen:
|
|
|
545 # print ("pos is greater")
|
|
|
546 pos = (pos - exonlen) - 1
|
|
|
547 # print ("new pos",pos)
|
|
|
548 else:
|
|
|
549 break
|
|
|
550 # print ("appending exon_start-pos", exon_start, pos, exon_start-pos)
|
|
|
551 genomic_pos_list.append((exon_start - pos) + 1)
|
|
|
552 return (chrom, genomic_pos_list)
|
|
|
553
|
|
|
554
|
|
|
555 orf_dict = {
|
|
|
556 "uorf": {},
|
|
|
557 "ouorf": {},
|
|
|
558 "cds": {},
|
|
|
559 "nested": {},
|
|
|
560 "odorf": {},
|
|
|
561 "dorf": {},
|
|
|
562 "minusone": {},
|
|
|
563 "readthrough": {},
|
|
|
564 "plusone": {},
|
|
|
565 "noncoding": {},
|
|
|
566 "extension": {},
|
|
|
567 "inframe_stop": {},
|
|
|
568 }
|
|
|
569
|
|
|
570 start_codons = ["ATG", "CTG", "GTG", "TTG", "ATC", "ATA", "ATT", "ACG", "AAG", "AGG"]
|
|
|
571
|
|
|
572 stop_codons = ["TAG", "TAA", "TGA"]
|
|
|
573
|
|
|
574
|
|
|
575 # Keep track of the longest transcript for each noncoding gene, append this to transcript list later
|
|
|
576 longest_noncoding = {}
|
|
|
577
|
|
|
578
|
|
|
579 tran_count = 0
|
|
|
580 # This section is used to gather all cds regions, convert them to genomic regions and store them in a dictionary to check against later (all transcript contribute to this not just those
|
|
|
581 # in the transcript list)
|
|
|
582 genomic_cds_dict = {}
|
|
|
583 tree_dict = {}
|
|
|
584 for transcript in master_dict:
|
|
|
585 # print (transcript, master_dict[transcript]["tran_type"])
|
|
|
586 tran_count += 1
|
|
|
587 if "seq" not in master_dict[transcript]:
|
|
|
588 continue
|
|
|
589 chrom = master_dict[transcript]["chrom"]
|
|
|
590 if chrom not in genomic_cds_dict:
|
|
|
591 genomic_cds_dict[chrom] = []
|
|
|
592 if "cds_start" in master_dict[transcript]:
|
|
|
593 cds_start = master_dict[transcript]["cds_start"]
|
|
|
594 cds_stop = master_dict[transcript]["cds_stop"]
|
|
|
595 if cds_start != "NULL":
|
|
|
596 cds_pos = []
|
|
|
597 for i in range(cds_start, cds_stop + 1):
|
|
|
598 cds_pos.append(i)
|
|
|
599
|
|
|
600 for tup in master_dict[transcript]["cds"]:
|
|
|
601 if tup[0] != tup[1]:
|
|
|
602 if tup not in genomic_cds_dict[chrom]:
|
|
|
603 genomic_cds_dict[chrom].append(tup)
|
|
|
604
|
|
|
605 print("genomic cds dict built")
|
|
|
606 print(list(genomic_cds_dict))
|
|
|
607 for chrom in genomic_cds_dict:
|
|
|
608 tree_dict[chrom] = IntervalTree.from_tuples(genomic_cds_dict[chrom])
|
|
|
609
|
|
|
610 # print (list(tree_dict))
|
|
|
611
|
|
|
612
|
|
|
613 connection = sqlite3.connect("{}.sqlite".format(organism))
|
|
|
614 cursor = connection.cursor()
|
|
|
615 cursor.execute(
|
|
|
616 "CREATE TABLE IF NOT EXISTS transcripts (transcript VARCHAR(50), gene VARCHAR(50), length INT(6), cds_start INT(6), cds_stop INT(6), sequence VARCHAR(50000), strand CHAR(1), stop_list VARCHAR(10000), start_list VARCHAR(10000), exon_junctions VARCHAR(1000), tran_type INT(1), gene_type INT(1), principal INT(1), version INT(2),gc INT(3),five_gc INT(3), cds_gc INT(3), three_gc INT(3), chrom VARCHAR(20));"
|
|
|
617 )
|
|
|
618 cursor.execute(
|
|
|
619 "CREATE TABLE IF NOT EXISTS coding_regions (transcript VARCHAR(50), coding_start INT(6), coding_stop INT(6));"
|
|
|
620 )
|
|
|
621 cursor.execute(
|
|
|
622 "CREATE TABLE IF NOT EXISTS exons (transcript VARCHAR(50), exon_start INT(6), exon_stop INT(6));"
|
|
|
623 )
|
|
|
624 cursor.execute(
|
|
|
625 "CREATE TABLE IF NOT EXISTS uorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
626 )
|
|
|
627 cursor.execute(
|
|
|
628 "CREATE TABLE IF NOT EXISTS ouorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
629 )
|
|
|
630 cursor.execute(
|
|
|
631 "CREATE TABLE IF NOT EXISTS cds (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
632 )
|
|
|
633 cursor.execute(
|
|
|
634 "CREATE TABLE IF NOT EXISTS nested (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
635 )
|
|
|
636 cursor.execute(
|
|
|
637 "CREATE TABLE IF NOT EXISTS odorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
638 )
|
|
|
639 cursor.execute(
|
|
|
640 "CREATE TABLE IF NOT EXISTS dorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
641 )
|
|
|
642 cursor.execute(
|
|
|
643 "CREATE TABLE IF NOT EXISTS minusone(transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
644 )
|
|
|
645 cursor.execute(
|
|
|
646 "CREATE TABLE IF NOT EXISTS readthrough (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
647 )
|
|
|
648 cursor.execute(
|
|
|
649 "CREATE TABLE IF NOT EXISTS plusone (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
650 )
|
|
|
651 cursor.execute(
|
|
|
652 "CREATE TABLE IF NOT EXISTS noncoding (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
653 )
|
|
|
654 cursor.execute(
|
|
|
655 "CREATE TABLE IF NOT EXISTS extension (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
656 )
|
|
|
657 cursor.execute(
|
|
|
658 "CREATE TABLE IF NOT EXISTS inframe_stop (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), cds_coverage FLOAT(20));"
|
|
|
659 )
|
|
|
660 connection.commit()
|
|
|
661
|
|
|
662
|
|
|
663 print("Finding ORFs")
|
|
|
664 transcript_count = 0
|
|
|
665 total_transcripts = len(list(master_dict))
|
|
|
666 for transcript in master_dict:
|
|
|
667 # print ("transcript",transcript)
|
|
|
668 # if transcript != "ENST00000316448":
|
|
|
669 # continue
|
|
|
670 transcript_count += 1
|
|
|
671 if transcript_count % 100 == 0:
|
|
|
672 print("Transcripts complete: {}/{}".format(transcript_count, total_transcripts))
|
|
|
673 if "seq" not in master_dict[transcript]:
|
|
|
674 print("transcript {} has no sequence".format(transcript))
|
|
|
675 continue
|
|
|
676 seq = master_dict[transcript]["seq"]
|
|
|
677 cds_start = "NULL"
|
|
|
678 cds_stop = "NULL"
|
|
|
679 transcript_len = len(seq)
|
|
|
680 if "cds_start" in master_dict[transcript]:
|
|
|
681 cds_start = master_dict[transcript]["cds_start"]
|
|
|
682 cds_stop = master_dict[transcript]["cds_stop"]
|
|
|
683
|
|
|
684 # Find out what regions of this transcript overlap with any other coding regions
|
|
|
685 coding_positions = []
|
|
|
686 if cds_start != "NULL":
|
|
|
687 # If this is a coding transcript don't bother checking the CDS
|
|
|
688 for i in range(cds_start, cds_stop):
|
|
|
689 coding_positions.append(i)
|
|
|
690 # check 5' leader
|
|
|
691 chrom, pos_list = tran_to_genome(transcript, 0, cds_start, master_dict)
|
|
|
692 for i in range(0, cds_start):
|
|
|
693 genomic_pos = pos_list[i]
|
|
|
694 overlap = tree_dict[chrom][genomic_pos]
|
|
|
695 if len(overlap) != 0:
|
|
|
696 coding_positions.append(i)
|
|
|
697 # check 3' trailer
|
|
|
698 chrom, pos_list = tran_to_genome(
|
|
|
699 transcript, cds_stop, transcript_len, master_dict
|
|
|
700 )
|
|
|
701 for i in range(cds_stop, transcript_len + 1):
|
|
|
702 # print ("i",i)
|
|
|
703 genomic_pos = pos_list[i - cds_stop]
|
|
|
704 # print ("genomic position",genomic_pos)
|
|
|
705 overlap = tree_dict[chrom][genomic_pos]
|
|
|
706 if len(overlap) != 0:
|
|
|
707 # print ("overlap not empty appending i",overlap)
|
|
|
708 coding_positions.append(i)
|
|
|
709 else:
|
|
|
710 # check entire transcript
|
|
|
711 chrom, pos_list = tran_to_genome(transcript, 0, transcript_len, master_dict)
|
|
|
712 for i in range(0, transcript_len):
|
|
|
713 genomic_pos = pos_list[i]
|
|
|
714 overlap = tree_dict[chrom][genomic_pos]
|
|
|
715 if len(overlap) != 0:
|
|
|
716 coding_positions.append(i)
|
|
|
717 coding_positions_tuple = to_ranges(coding_positions)
|
|
|
718 coding_dict[transcript] = coding_positions_tuple
|
|
|
719 coding_positions = set(coding_positions)
|
|
|
720 # if this is a coding transcript find the minusone, readhtrough, plusone co-ordinates
|
|
|
721 if cds_start != "NULL":
|
|
|
722 # print ("transcript", transcript)
|
|
|
723 # if pseudo_utr_len != 0:
|
|
|
724 # cds_stop -= 3 # take 3 from stop so we can match it with orf_stop, do it here rather than above in case cds_stop is null
|
|
|
725 recoding_dict = {2: "minusone", 0: "readthrough", 1: "plusone"}
|
|
|
726 for addition in recoding_dict:
|
|
|
727 orftype = recoding_dict[addition]
|
|
|
728 for i in range(cds_stop + addition, transcript_len, 3):
|
|
|
729 if seq[i : i + 3] in stop_codons:
|
|
|
730 # orf_seq = seq[cds_stop:i+3]
|
|
|
731 orf_start = cds_stop
|
|
|
732 if orftype == "readthrough":
|
|
|
733 orf_start -= 2
|
|
|
734 if orftype == "plusone":
|
|
|
735 orf_start -= 1
|
|
|
736 orf_stop = i + 3 # +2 so it refers to the end of the stop codon
|
|
|
737 start_codon = None
|
|
|
738 length = (i + 3) - cds_stop
|
|
|
739 orf_pos_list = []
|
|
|
740 # determine how many nucleotides in this orf overlap with an annotated coding region
|
|
|
741 cds_cov_count = 0.0
|
|
|
742 for position in range(orf_start, orf_stop):
|
|
|
743 orf_pos_list.append(position)
|
|
|
744 for pos in range(orf_start, orf_stop + 1):
|
|
|
745 if pos in coding_positions:
|
|
|
746 cds_cov_count += 1
|
|
|
747 cds_cov = cds_cov_count / length
|
|
|
748 # print ("orftype, start, stop", orftype, orf_start, orf_stop)
|
|
|
749 cursor.execute(
|
|
|
750 "INSERT INTO {} VALUES('{}','{}',{},{},{},{});".format(
|
|
|
751 orftype,
|
|
|
752 transcript,
|
|
|
753 start_codon,
|
|
|
754 length,
|
|
|
755 orf_start,
|
|
|
756 orf_stop,
|
|
|
757 cds_cov,
|
|
|
758 )
|
|
|
759 )
|
|
|
760 break
|
|
|
761 # sys.exit()
|
|
|
762 for frame in [0, 1, 2]:
|
|
|
763 for i in range(frame, transcript_len, 3):
|
|
|
764 if seq[i : i + 3] in start_codons:
|
|
|
765 for x in range(i, transcript_len, 3):
|
|
|
766 if seq[x : x + 3] in stop_codons:
|
|
|
767 # orf_seq = seq[i:x+3]
|
|
|
768 orf_start = i + 1
|
|
|
769 orf_stop = x + 3 # +2 so it refers to the end of the stop codon
|
|
|
770 start_codon = seq[i : i + 3]
|
|
|
771 length = (x + 3) - i
|
|
|
772 orf_pos_list = []
|
|
|
773 # determine how many nucleotides in this orf overlap with an annotated coding region
|
|
|
774 cds_cov_count = 0.0
|
|
|
775 for pos in range(orf_start, orf_stop + 1):
|
|
|
776 if pos in coding_positions:
|
|
|
777 cds_cov_count += 1
|
|
|
778 cds_cov = float(cds_cov_count) / float(length)
|
|
|
779 # Now determine orf type
|
|
|
780 if cds_start == "NULL":
|
|
|
781 orftype = "noncoding"
|
|
|
782 else:
|
|
|
783 # print ("cds start is not null :{}:{}".format(cds_start,cds_stop))
|
|
|
784 # print ("orf start, orf stop", orf_start, orf_stop)
|
|
|
785 if orf_start == cds_start and orf_stop == cds_stop:
|
|
|
786 orftype = "cds"
|
|
|
787 # print ("orf type is cds")
|
|
|
788 elif orf_start < cds_start and orf_stop == cds_stop:
|
|
|
789 orftype = "extension"
|
|
|
790 # special case for extensions, we only take from the orf_start to the cds_start, and re-calculate cds coverage
|
|
|
791 orf_stop = cds_start
|
|
|
792 cds_cov_count = 0.0
|
|
|
793 for pos in range(orf_start, orf_stop + 1):
|
|
|
794 if pos in coding_positions:
|
|
|
795 cds_cov_count += 1
|
|
|
796 cds_cov = float(cds_cov_count) / float(length)
|
|
|
797 # orf_seq = seq[orf_start:cds_start]
|
|
|
798 length = cds_start - orf_start
|
|
|
799 # print ("orf type is extension")
|
|
|
800 elif orf_start < cds_start and orf_stop <= cds_start:
|
|
|
801 orftype = "uorf"
|
|
|
802 # print ("orf type is uorf")
|
|
|
803 elif orf_start < cds_start and orf_stop > cds_start:
|
|
|
804 orftype = "ouorf"
|
|
|
805 # print ("orf type is ouorf")
|
|
|
806 # sys.exit()
|
|
|
807 elif (
|
|
|
808 orf_start >= cds_start
|
|
|
809 and orf_start <= cds_stop
|
|
|
810 and orf_stop <= cds_stop
|
|
|
811 ):
|
|
|
812 if orf_stop == cds_stop:
|
|
|
813 break
|
|
|
814 # print ("Tran, cds_start, cds_stop, orf_start, orf_stop, tranlen",tran, cds_start, cds_stop, orf_start, orf_stop,transcript_len)
|
|
|
815 if (
|
|
|
816 orf_stop < transcript_len
|
|
|
817 and orf_stop % 3 == cds_stop % 3
|
|
|
818 ) or (
|
|
|
819 cds_start != 1
|
|
|
820 and orf_stop % 3 == (cds_start + 2) % 3
|
|
|
821 ):
|
|
|
822 # print ("Transcript {} has an inframe stop codon".format(transcript))
|
|
|
823 break
|
|
|
824 orftype = "nested"
|
|
|
825 # print ("orf type is nested")
|
|
|
826 elif (
|
|
|
827 orf_start >= cds_start
|
|
|
828 and orf_start <= cds_stop
|
|
|
829 and orf_stop > cds_stop
|
|
|
830 ):
|
|
|
831 orftype = "odorf"
|
|
|
832 # print ("orftype is odorf")
|
|
|
833 elif orf_start > cds_stop and orf_stop > cds_stop:
|
|
|
834 orftype = "dorf"
|
|
|
835 # print ("orftype is dorf")
|
|
|
836 # if orf_stop > cds_start and orf_stop < cds_stop:
|
|
|
837 # if (orf_stop+1)%3 == cds_start%3:
|
|
|
838 # orftype = "inframe_stop"
|
|
|
839 # print ("inframe stop, transcript, orf_stop", transcript, orf_stop)
|
|
|
840 # sys.exit()
|
|
|
841 # if transcript not in orf_dict:
|
|
|
842 # orf_dict[orftype][transcript] = []
|
|
|
843 # #print ("some weird stop or something")
|
|
|
844 cursor.execute(
|
|
|
845 "INSERT INTO {} VALUES('{}','{}',{},{},{},{});".format(
|
|
|
846 orftype,
|
|
|
847 transcript,
|
|
|
848 start_codon,
|
|
|
849 length,
|
|
|
850 orf_start,
|
|
|
851 orf_stop,
|
|
|
852 cds_cov,
|
|
|
853 )
|
|
|
854 )
|
|
|
855 break
|
|
|
856 # Used to keep track of the codons at cds_start and cds_stop positions,
|
|
|
857 # If there is an issue with the cds co-ordinates the starts and stops counts will
|
|
|
858 # be much lower than the other count, start with 1 to prevent division by 0
|
|
|
859 nuc_dict = {"stops": {"stops": 1, "other": 0}, "starts": {"starts": 1, "other": 0}}
|
|
|
860
|
|
|
861
|
|
|
862 def calcgc(seq):
|
|
|
863 if seq == "":
|
|
|
864 return "NULL"
|
|
|
865 g_count = 0
|
|
|
866 c_count = 0
|
|
|
867 a_count = 0
|
|
|
868 t_count = 0
|
|
|
869 for char in seq:
|
|
|
870 if char == "A":
|
|
|
871 a_count += 1
|
|
|
872 if char == "T":
|
|
|
873 t_count += 1
|
|
|
874 if char == "G":
|
|
|
875 g_count += 1
|
|
|
876 if char == "C":
|
|
|
877 c_count += 1
|
|
|
878 gc = ((g_count + c_count) / float(len(seq))) * 100
|
|
|
879 return round(gc, 2)
|
|
|
880
|
|
|
881
|
|
|
882 for transcript in master_dict:
|
|
|
883 # print ("transcripts", transcript)
|
|
|
884 length = master_dict[transcript]["length"]
|
|
|
885 cds_start = master_dict[transcript]["cds_start"]
|
|
|
886 cds_stop = master_dict[transcript]["cds_stop"]
|
|
|
887 seq = master_dict[transcript]["seq"]
|
|
|
888 strand = master_dict[transcript]["strand"]
|
|
|
889 chrom = master_dict[transcript]["chrom"]
|
|
|
890 gene = master_dict[transcript]["gene_name"]
|
|
|
891 gc = calcgc(seq)
|
|
|
892 five_gc = "NULL"
|
|
|
893 cds_gc = "NULL"
|
|
|
894 three_gc = "NULL"
|
|
|
895 if cds_start != "NULL":
|
|
|
896 five_gc = calcgc(seq[:cds_start])
|
|
|
897 cds_gc = calcgc(seq[cds_start:cds_stop])
|
|
|
898 three_gc = calcgc(seq[cds_stop:])
|
|
|
899 # check that the nucleotide cds_start points to is the first of the start codon
|
|
|
900 # take one becase cds_start is 1 based but python indexing is 0 based
|
|
|
901 start_nuc = seq[cds_start - 1 : cds_start + 2]
|
|
|
902 # print ("start nuc",start_nuc)
|
|
|
903 if start_nuc == "ATG":
|
|
|
904 nuc_dict["starts"]["starts"] += 1
|
|
|
905 else:
|
|
|
906 nuc_dict["starts"]["other"] += 1
|
|
|
907 stop_nuc = seq[cds_stop - 3 : cds_stop]
|
|
|
908 # print ("stop_nuc",stop_nuc)
|
|
|
909 if stop_nuc in ["TAG", "TAA", "TGA"]:
|
|
|
910 nuc_dict["stops"]["stops"] += 1
|
|
|
911 else:
|
|
|
912 nuc_dict["stops"]["other"] += 1
|
|
|
913 tran_type = master_dict[transcript]["tran_type"]
|
|
|
914 if coding_genes_dict[gene] == True:
|
|
|
915 gene_type = 1
|
|
|
916 else:
|
|
|
917 gene_type = 0
|
|
|
918 # print ("tran type before",tran_type)
|
|
|
919 if tran_type == "coding":
|
|
|
920 tran_type = 1
|
|
|
921 else:
|
|
|
922 tran_type = 0
|
|
|
923 # print ("tran type after",tran_type)
|
|
|
924 start_list = str(master_dict[transcript]["start_list"]).replace(" ", "").strip("[]")
|
|
|
925 stop_list = str(master_dict[transcript]["stop_list"]).replace(" ", "").strip("[]")
|
|
|
926 exon_junctions = (
|
|
|
927 str(master_dict[transcript]["exon_junctions"]).replace(" ", "").strip("[]")
|
|
|
928 )
|
|
|
929 principal = master_dict[transcript]["principal"]
|
|
|
930 version = master_dict[transcript]["version"]
|
|
|
931 # print (master_dict[transcript])
|
|
|
932 # print (tran_type)
|
|
|
933 # print (gene_type)
|
|
|
934 # print (principal)
|
|
|
935 # print (version)
|
|
|
936 # print ("INSERT INTO transcripts VALUES('{}','{}',{},{},{},'{}','{}','{}','{}','{}',{},{},{},{});".format(transcript, gene, length, cds_start, cds_stop, seq, strand,stop_list, start_list, exon_junctions, tran_type,gene_type,principal,version))
|
|
|
937 cursor.execute(
|
|
|
938 "INSERT INTO transcripts VALUES('{}','{}',{},{},{},'{}','{}','{}','{}','{}',{},{},{},{},{},{},{},{},'{}');".format(
|
|
|
939 transcript,
|
|
|
940 gene,
|
|
|
941 length,
|
|
|
942 cds_start,
|
|
|
943 cds_stop,
|
|
|
944 seq,
|
|
|
945 strand,
|
|
|
946 stop_list,
|
|
|
947 start_list,
|
|
|
948 exon_junctions,
|
|
|
949 tran_type,
|
|
|
950 gene_type,
|
|
|
951 principal,
|
|
|
952 version,
|
|
|
953 gc,
|
|
|
954 five_gc,
|
|
|
955 cds_gc,
|
|
|
956 three_gc,
|
|
|
957 chrom,
|
|
|
958 )
|
|
|
959 )
|
|
|
960
|
|
|
961 for tup in master_dict[transcript]["exon"]:
|
|
|
962 cursor.execute(
|
|
|
963 "INSERT INTO exons VALUES('{}',{},{});".format(transcript, tup[0], tup[1])
|
|
|
964 )
|
|
|
965 if transcript in coding_dict:
|
|
|
966 for tup in coding_dict[transcript]:
|
|
|
967 cursor.execute(
|
|
|
968 "INSERT INTO coding_regions VALUES('{}',{},{});".format(
|
|
|
969 transcript, tup[0], tup[1]
|
|
|
970 )
|
|
|
971 )
|
|
|
972
|
|
|
973 connection.commit()
|
|
|
974 connection.close()
|
|
|
975
|
|
|
976 print("delim", delimiters)
|
|
|
977 if (nuc_dict["starts"]["other"] / nuc_dict["starts"]["starts"]) > 0.05:
|
|
|
978 print(
|
|
|
979 "Warning: {} transcripts do not have a an AUG at the CDS start position".format(
|
|
|
980 nuc_dict["starts"]["other"]
|
|
|
981 )
|
|
|
982 )
|
|
|
983 if (nuc_dict["stops"]["other"] / nuc_dict["stops"]["stops"]) > 0.05:
|
|
|
984 print(
|
|
|
985 "Warning: {} transcripts do not have a an stop codon at the CDS stop position".format(
|
|
|
986 nuc_dict["stops"]["other"]
|
|
|
987 )
|
|
|
988 )
|
|
|
989 if len(notinannotation) > 0:
|
|
|
990 print(
|
|
|
991 "Warning: {} transcripts were in the fasta file, but not the annotation file, these will be discarded".format(
|
|
|
992 len(notinannotation)
|
|
|
993 )
|
|
|
994 )
|
