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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 import intervaltree
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10 from intervaltree import Interval, IntervalTree
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11 import itertools
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12
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13
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14
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15 #This should be a GTF or GFF3 file
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16 annotation_file = open(sys.argv[1],"r")
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17 #This needs to be the transcriptomic fasta file
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18 fasta_file = open(sys.argv[2],"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[3])
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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[4]
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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[5]
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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
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30 delimiters = {"transcripts":{"before":[],"after":[],"annot_types": ["cds","utr"]},
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31 "genes":{"before":[],"after":['"'],"annot_types": ["lnc_rna"]}}
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32
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33 punctuation = [";"," ","-",":","-",".","=","\t"]
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34 #Find delimiters in the annotation and fasta files using the user_transcript_id
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35 #and user_gene_name examples given by user.
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36 for line in annotation_file:
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37 if user_transcript_id in line:
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38 tabsplitline = line.split("\t")
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39 annot_type = tabsplitline[2]
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40 if annot_type not in delimiters["transcripts"]["annot_types"]:
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41 delimiters["transcripts"]["annot_types"].append(annot_type.lower())
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42 splitline = line.split(user_transcript_id)
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43 before_delimiter = splitline[0]
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44 for item in punctuation:
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45 if item in before_delimiter:
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46 if len(before_delimiter.split(item)[-1]) >= 5:
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47 before_delimiter = before_delimiter.split(item)[-1]
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48 after_delimiter = splitline[1][:2]
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49 if before_delimiter not in delimiters["transcripts"]["before"] and len(before_delimiter) >= 5:
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50 delimiters["transcripts"]["before"].append(before_delimiter)
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51 if after_delimiter not in delimiters["transcripts"]["after"]:
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52 delimiters["transcripts"]["after"].append(after_delimiter)
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53 if user_gene_name in line:
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54 tabsplitline = line.split("\t")
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55 annot_type = tabsplitline[2]
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56 if annot_type not in delimiters["genes"]["annot_types"]:
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57 delimiters["genes"]["annot_types"].append(annot_type.lower())
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58 splitline = line.split(user_gene_name)
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59 before_delimiter = splitline[0]
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60 for item in punctuation:
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61 if item in before_delimiter:
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62 if len(before_delimiter.split(item)[-1]) >= 5:
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63 before_delimiter = before_delimiter.split(item)[-1]
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64 after_delimiter = splitline[1][0]
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65 if before_delimiter not in delimiters["genes"]["before"] and len(before_delimiter) >= 5:
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66 delimiters["genes"]["before"].append(before_delimiter)
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67 if after_delimiter not in delimiters["genes"]["after"]:
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68 if after_delimiter in punctuation:
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69 delimiters["genes"]["after"].append(after_delimiter)
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70 for line in fasta_file:
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71 if user_transcript_id in line:
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72 splitline = line.split(user_transcript_id)
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73 before_delimiter = splitline[0]
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74 for item in punctuation:
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75 if item in before_delimiter:
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76 if len(before_delimiter.split(item)[1]) >= 5:
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77 before_delimiter = before_delimiter.split(item)[1]
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78 after_delimiter = splitline[1][0]
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79 if before_delimiter not in delimiters["transcripts"]["before"] and len(before_delimiter) >= 5:
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80 delimiters["transcripts"]["before"].append(before_delimiter)
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81 if after_delimiter not in delimiters["transcripts"]["after"]:
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82 delimiters["transcripts"]["after"].append(after_delimiter)
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83 fasta_file.close()
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84 annotation_file.close()
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85
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86
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87
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88
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89
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90 if delimiters["transcripts"]["before"] == []:
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91 print ("ERROR: No transcript_id with the name {} could be found in the annotation file".format(user_transcript_id))
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92 sys.exit()
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93 if delimiters["genes"]["before"] == []:
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94 print ("ERROR: No gene with the name {} could be found in the annotation file".format(user_gene_name))
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95 sys.exit()
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96
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97 master_dict = {}
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98 coding_dict = {}
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99 notinfasta = open("notinfasta.csv","w")
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100
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101 #Given a nucleotide sequence returns the positions of all start and stop codons.
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102 def get_start_stops(transcript_sequence):
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103 transcript_sequence = transcript_sequence.upper()
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104 start_codons = ['ATG']
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105 stop_codons = ['TAA', 'TAG', 'TGA']
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106 seq_frames = {'starts': [], 'stops': []}
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107 for codons, positions in ((start_codons, 'starts'),(stop_codons, 'stops')):
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108 if len(codons) > 1:
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109 pat = re.compile('|'.join(codons))
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110 else:
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111 pat = re.compile(codons[0])
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112 for m in re.finditer(pat, transcript_sequence):
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113 # Increment position by 1, Frame 1 starts at position 1 not 0,
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114 # if it's a stop codon add another 2 so it points to the last nuc of the codon
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115 if positions == "starts":
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116 start = m.start() + 1
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117 else:
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118 start = m.start() + 3
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119 seq_frames[positions].append(start)
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120 return seq_frames
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121
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122
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123 #parse fasta to get the nucleotide sequence of transcripts and the positions of start/stop codons.
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124 fasta_file = open(sys.argv[2],"r")
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125 read_fasta = fasta_file.read()
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126 split_fasta = read_fasta.split(">")
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127 for entry in split_fasta[1:]:
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128 newline_split = entry.split("\n")
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129 tran = newline_split[0]
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130 for item in delimiters["transcripts"]["after"]:
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131 if item in tran:
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132 tran = tran.split(item)[0]
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133 tran = tran.replace("-","_").replace("(","").replace(")","")
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134 seq = ("".join(newline_split[1:]))
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135 if "_PAR_Y" in tran:
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136 tran += "_chrY"
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137 elif "_PAR_X" in tran:
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138 tran += "_chrX"
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139 tran = tran.upper()
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140 starts_stops = get_start_stops(seq)
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141 if tran not in master_dict:
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142 master_dict[tran] = {"utr":[], "cds":[], "exon":[],"start_codon":[],"stop_codon":[],"start_list":starts_stops["starts"],
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143 "stop_list":starts_stops["stops"],"transcript":[], "strand":"" ,"gene_name":"","chrom":"","seq":seq,"cds_start":"NULL","cds_stop":"NULL",
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144 "length":len(seq),"principal":0,"version":"NULL"}
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145
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146
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147
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148
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149 def to_ranges(iterable):
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150 tup_list = []
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151 iterable = sorted(set(iterable))
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152 for key, group in itertools.groupby(enumerate(iterable),lambda t: t[1] - t[0]):
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153 group = list(group)
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154 tup_list.append((group[0][1], group[-1][1]))
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155 return tup_list
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156
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157 #parse annotation file to get chromsome, exon location and CDS info for each transcript
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158 def parse_gtf_file(annotation_file):
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159 for line in annotation_file:
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160 if line == "\n":
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161 continue
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162 if line[0] != '#':
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163 splitline = (line.replace("\n","")).split("\t")
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164 chrom = splitline[0]
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165 try:
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166 annot_type = splitline[2].lower()
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167 except:
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168 print ("ERROR tried to index to second item in splitline: ",splitline, line)
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169 sys.exit()
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170 #if annot_type not in ["cds", "utr", "exon", "transcript","five_prime_utr", "three_prime_utr","stop_codon","start_codon"]:
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171 # continue
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172 if annot_type not in delimiters["transcripts"]["annot_types"] and annot_type not in delimiters["genes"]["annot_types"]:
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173 continue
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174 if annot_type == "five_prime_utr" or annot_type == "three_prime_utr":
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175 annot_type = "utr"
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176 strand = splitline[6]
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177 if strand == "+":
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178 start = int(splitline[3])
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179 end = int(splitline[4])
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180 else:
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181 start = int(splitline[3])+1
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182 end = int(splitline[4])+1
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183 desc = splitline[8]
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184 tran = desc
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185 gene = desc
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186 for item in delimiters["transcripts"]["before"]:
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187 if item in tran:
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188 tran = tran.split(item)[1]
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189 for item in delimiters["transcripts"]["after"]:
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190 if item in tran:
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191 tran = tran.split(item)[0]
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192 if "." in tran and TRAN_VERSION == True:
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193 #print ("raw tran",tran)
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194 tran = tran.split(".")
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195 version = int(tran[-1].split("_")[0])
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196 tran = tran[0]
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197 else:
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198 version = "NULL"
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199 tran = tran.replace("-","_").replace(".","_")
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200 tran = tran.replace("(","").replace(")","")
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201 tran = tran.replace(" ","").replace("\t","")
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202 tran = tran.upper()
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203 tran = tran.replace("GENE_","").replace("ID_","")
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204 #print ("tran",tran,version)
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205 #if tran == "ENST00000316448":
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206 # print ("annot type",annot_type)
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207 # print ("appending exon to tran", start, end,line)
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208
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209 gene_found = False
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210
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211 if annot_type in delimiters["genes"]["annot_types"]:
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212 for item in delimiters["genes"]["before"]:
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213 if item in gene:
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214 gene_found = True
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215 gene = gene.split(item)[1]
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216 for item in delimiters["genes"]["after"]:
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217 if item in gene:
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218 gene = gene.split(item)[0]
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219 gene = gene.replace("'","''")
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220 gene = gene.replace("GENE_","")
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221 gene = gene.replace("ID_","")
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222 gene = gene.upper()
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223
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224 if tran in master_dict:
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225 if annot_type in master_dict[tran]:
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226 master_dict[tran][annot_type].append((start, end))
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227 master_dict[tran]["strand"] = strand
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228 master_dict[tran]["chrom"] = chrom
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229 master_dict[tran]["version"] = version
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230 if gene_found == True:
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231 master_dict[tran]["gene_name"] = gene
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232 else:
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233 notinfasta.write("{}\n".format(tran))
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234
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235 annotation_file = open(sys.argv[1],"r")
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236 parse_gtf_file(annotation_file)
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237
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238
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239 #remove transcripts that were in fasta file but not in annotation_file
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240 notinannotation = []
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241 for tran in master_dict:
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242 if master_dict[tran]["chrom"] == "":
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243 #print ("tran {} has no chrom :(".format(tran))
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244 notinannotation.append(tran)
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245 for tran in notinannotation:
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246 del master_dict[tran]
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247
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248 #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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249 coding_genes_dict = {}
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250 #parse master_dict to calculate length, cds_start/stop and exon junction positions
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251 for tran in master_dict:
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252 try:
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253 transeq = master_dict[tran]["seq"]
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254 except Exception as e:
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255 print ("not in fasta", tran)
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256 notinfasta.write("{}\n".format(tran))
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257 continue
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258 exon_junctions = []
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259 total_length = len(transeq)
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260 three_len = 1
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261 five_len = 1
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262 strand = master_dict[tran]["strand"]
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263 if master_dict[tran]["gene_name"] == "":
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264 master_dict[tran]["gene_name"] = tran
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265 gene = master_dict[tran]["gene_name"]
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266 if gene not in coding_genes_dict:
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267 coding_genes_dict[gene] = False
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268
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269 if master_dict[tran]["cds"] == []:
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270 tran_type = "noncoding"
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271 cds_start = 'NULL'
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272 cds_stop = 'NULL'
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273 else:
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274 #get utr lengths from annotation
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275 tran_type = "coding"
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276 coding_genes_dict[gene] = True
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277 sorted_exons = sorted(master_dict[tran]["exon"])
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278 sorted_cds = sorted(master_dict[tran]["cds"])
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279 min_cds = sorted_cds[0][0]
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280 #Some annotation files do not have utr annotation types, so fix that here if thats the case
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281 if master_dict[tran]["utr"] == []:
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282 for exon_tup in master_dict[tran]["exon"]:
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283 if exon_tup not in master_dict[tran]["cds"]:
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284 # Now check if this overlaps with any of the CDS exons
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285 overlap = False
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286 for cds_tup in master_dict[tran]["cds"]:
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287 if exon_tup[0] == cds_tup[0] and exon_tup[1] != cds_tup[1]:
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288 master_dict[tran]["utr"].append((cds_tup[1],exon_tup[1]))
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289 overlap = True
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290 if exon_tup[0] != cds_tup[0] and exon_tup[1] == cds_tup[1]:
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291 master_dict[tran]["utr"].append((exon_tup[0],cds_tup[0]))
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292 overlap = True
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293 if overlap == False:
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294 master_dict[tran]["utr"].append(exon_tup)
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295
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296
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297 '''
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298 if tran == "NM_001258497":
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299 print ("sorted cds",sorted_cds)
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300 print ("min cds",min_cds)
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301 print ("chrom",master_dict[tran]["chrom"])
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302 print ("sorted exons", sorted_exons)
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303 print ("utr",master_dict[tran]["utr"])
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304 sys.exit()
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305 '''
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306 #if tran == "ENST00000381401":
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307 # print ("min cds,sorted utr",min_cds,sorted(master_dict[tran]["utr"]))
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308 for tup in sorted(master_dict[tran]["utr"]):
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309 #if tran == "ENST00000381401":
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310 # print ("tup", tup)
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311 if tup[0] < min_cds:
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312 five_len += (tup[1]-tup[0])+1
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313 #if tran == "ENST00000381401":
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314 # print ("adding to fivelen")
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315 elif tup[0] > min_cds:
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316 three_len += (tup[1] - tup[0])+1
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317 #if tran == "ENST00000381401":
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318 # print ("adding to three len")
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319 else:
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320 pass
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321 if strand == "+":
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322 if len(sorted_exons) > 1:
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323 sorted_exons[0] = (sorted_exons[0][0]-pseudo_utr_len, sorted_exons[0][1])
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324 sorted_exons[-1] = (sorted_exons[-1][0], sorted_exons[-1][1]+pseudo_utr_len)
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325 else:
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326 sorted_exons[0] = (sorted_exons[0][0]-pseudo_utr_len, sorted_exons[0][1]+pseudo_utr_len)
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327 master_dict[tran]["exon"] = sorted_exons
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328 cds_start = (five_len+pseudo_utr_len)
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329 cds_stop = ((total_length - three_len)-pseudo_utr_len)+1
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330 elif strand == "-":
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331 if len(sorted_exons) > 1:
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332 sorted_exons[0] = (sorted_exons[0][0]-pseudo_utr_len, sorted_exons[0][1])
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333 sorted_exons[-1] = (sorted_exons[-1][0], sorted_exons[-1][1]+pseudo_utr_len)
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334 else:
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335 sorted_exons[0] = (sorted_exons[0][0]-pseudo_utr_len, sorted_exons[0][1]+pseudo_utr_len)
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336 master_dict[tran]["exon"] = sorted_exons
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337 cds_start = (three_len+pseudo_utr_len)
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338 cds_stop = ((total_length - (five_len))-pseudo_utr_len)+1
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339 #if tran == "ENST00000381401":
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340 # print ("cds start, cds stop, five_len, three_len",cds_start,cds_stop,five_len,three_len)
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341 # #sys.exit()
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342 else:
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343 print ("strand is unknown: {}".format(strand))
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344 sys.exit()
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345
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346 #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
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|
|
347 total_intronic = 0
|
|
|
348 try:
|
|
|
349 if strand == "+":
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|
350 tx_start = min(sorted(master_dict[tran]["exon"]))[0]
|
|
|
351 prev_end = tx_start
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|
|
352 for tup in sorted(master_dict[tran]["exon"])[:-1]:
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|
|
353 total_intronic += tup[0]-prev_end
|
|
|
354 exon_junctions.append(((tup[1])-tx_start)-total_intronic)
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|
|
355 prev_end = tup[1]
|
|
|
356 elif strand == "-":
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|
|
357 tx_start = max(sorted(master_dict[tran]["exon"]))[-1]
|
|
|
358 prev_end = tx_start
|
|
|
359 for tup in (sorted(master_dict[tran]["exon"])[1:])[::-1]:
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|
|
360 total_intronic += (tup[0]+1)-prev_end
|
|
|
361 exon_junctions.append(((tup[1])-tx_start)-total_intronic)
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|
|
362 prev_end = tup[1]
|
|
|
363 except:
|
|
|
364 if strand == "+":
|
|
|
365 tx_start = min(sorted(master_dict[tran]["cds"]))[0]
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|
|
366 prev_end = tx_start
|
|
|
367 for tup in sorted(master_dict[tran]["cds"])[:-1]:
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|
|
368 total_intronic += tup[0]-prev_end
|
|
|
369 exon_junctions.append(((tup[1])-tx_start)-total_intronic)
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370 prev_end = tup[1]
|
|
|
371 elif strand == "-":
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|
|
372 tx_start = max(sorted(master_dict[tran]["cds"]))[-1]
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|
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373 prev_end = tx_start
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|
|
374 for tup in (sorted(master_dict[tran]["cds"])[1:])[::-1]:
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|
|
375 total_intronic += (tup[0]+1)-prev_end
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|
376 exon_junctions.append(((tup[1])-tx_start)-total_intronic)
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|
|
377 prev_end = tup[1]
|
|
|
378 if strand == "+" and cds_start != "NULL":
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|
|
379 master_dict[tran]["cds_start"] = cds_start
|
|
|
380 master_dict[tran]["cds_stop"] = cds_stop
|
|
|
381 elif strand == "-" and cds_start != "NULL":
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|
|
382 master_dict[tran]["cds_start"] = cds_start
|
|
|
383 master_dict[tran]["cds_stop"] = cds_stop
|
|
|
384 master_dict[tran]["strand"] = strand
|
|
|
385 master_dict[tran]["tran_type"] = tran_type
|
|
|
386 master_dict[tran]["exon_junctions"] = exon_junctions
|
|
|
387
|
|
|
388 longest_tran_dict = {}
|
|
|
389 for tran in master_dict:
|
|
|
390 try:
|
|
|
391 gene = master_dict[tran]["gene_name"]
|
|
|
392 except:
|
|
|
393 continue
|
|
|
394 if coding_genes_dict[gene] == True:
|
|
|
395 if "cds_start" in master_dict[tran]:
|
|
|
396 if master_dict[tran]["cds_stop"] != "NULL" and master_dict[tran]["cds_start"] != "NULL":
|
|
|
397 cds_length = master_dict[tran]["cds_stop"]- master_dict[tran]["cds_start"]
|
|
|
398 if gene not in longest_tran_dict:
|
|
|
399 longest_tran_dict[gene] = {"tran":tran,"length":cds_length}
|
|
|
400 else:
|
|
|
401 if cds_length > longest_tran_dict[gene]["length"]:
|
|
|
402 longest_tran_dict[gene] = {"tran":tran,"length":cds_length}
|
|
|
403 if cds_length == longest_tran_dict[gene]["length"]:
|
|
|
404 if master_dict[tran]["length"] > master_dict[longest_tran_dict[gene]["tran"]]["length"]:
|
|
|
405 longest_tran_dict[gene] = {"tran":tran,"length":cds_length}
|
|
|
406 else:
|
|
|
407 length = master_dict[tran]["length"]
|
|
|
408 if gene not in longest_tran_dict:
|
|
|
409 longest_tran_dict[gene] = {"tran":tran,"length":length}
|
|
|
410 elif length > longest_tran_dict[gene]["length"]:
|
|
|
411 longest_tran_dict[gene] = {"tran":tran,"length":length}
|
|
|
412
|
|
|
413
|
|
|
414
|
|
|
415
|
|
|
416
|
|
|
417 for gene in longest_tran_dict:
|
|
|
418 longest_tran = longest_tran_dict[gene]["tran"]
|
|
|
419 master_dict[longest_tran]["principal"] = 1
|
|
|
420
|
|
|
421 gene_sample = []
|
|
|
422 for key in list(master_dict)[:10]:
|
|
|
423 try:
|
|
|
424 gene_sample.append(master_dict[key]["gene_name"])
|
|
|
425 except:
|
|
|
426 pass
|
|
|
427
|
|
|
428 print ("Here is a sample of the transcript ids: {}".format(list(master_dict)[:10]))
|
|
|
429 print ("Here is a sample of the gene names: {}".format(gene_sample))
|
|
|
430
|
|
|
431
|
|
|
432 # 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.
|
|
|
433 def tran_to_genome(tran, start_pos, end_pos, master_dict):
|
|
|
434 pos_list = []
|
|
|
435 for i in range(start_pos,end_pos+1):
|
|
|
436 pos_list.append(i)
|
|
|
437 genomic_pos_list = []
|
|
|
438 if tran in master_dict:
|
|
|
439 transcript_info = master_dict[tran]
|
|
|
440 else:
|
|
|
441 return ("Null", [])
|
|
|
442
|
|
|
443 chrom = transcript_info["chrom"]
|
|
|
444 strand = transcript_info["strand"]
|
|
|
445 exons = transcript_info["exon"]
|
|
|
446 #print ("chrom,strand,exons",chrom,strand,exons)
|
|
|
447 for pos in pos_list:
|
|
|
448 #print ("pos",pos)
|
|
|
449 if strand == "+":
|
|
|
450 exon_start = 0
|
|
|
451 for tup in exons:
|
|
|
452 #print ("tup",tup)
|
|
|
453 exon_start = tup[0]
|
|
|
454 exonlen = tup[1] - tup[0]
|
|
|
455 if pos > exonlen:
|
|
|
456 pos = (pos - exonlen)-1
|
|
|
457 else:
|
|
|
458 break
|
|
|
459 #print ("appending exon_start-pos", exon_start, pos, exon_start+pos)
|
|
|
460 genomic_pos_list.append((exon_start+pos)-1)
|
|
|
461 elif strand == "-":
|
|
|
462 exon_start = 0
|
|
|
463 for tup in exons[::-1]:
|
|
|
464 #print ("tup",tup)
|
|
|
465 exon_start = tup[1]
|
|
|
466 exonlen = tup[1] - tup[0]
|
|
|
467 #print ("exonlen",exonlen)
|
|
|
468 if pos > exonlen:
|
|
|
469 #print ("pos is greater")
|
|
|
470 pos = (pos - exonlen)-1
|
|
|
471 #print ("new pos",pos)
|
|
|
472 else:
|
|
|
473 break
|
|
|
474 #print ("appending exon_start-pos", exon_start, pos, exon_start-pos)
|
|
|
475 genomic_pos_list.append((exon_start-pos)+1)
|
|
|
476 return (chrom, genomic_pos_list)
|
|
|
477
|
|
|
478
|
|
|
479
|
|
|
480
|
|
|
481 orf_dict = {"uorf":{},
|
|
|
482 "ouorf":{},
|
|
|
483 "cds":{},
|
|
|
484 "nested":{},
|
|
|
485 "odorf":{},
|
|
|
486 "dorf":{},
|
|
|
487 "minusone":{},
|
|
|
488 "readthrough":{},
|
|
|
489 "plusone":{},
|
|
|
490 "noncoding":{},
|
|
|
491 "extension":{},
|
|
|
492 "inframe_stop":{}
|
|
|
493 }
|
|
|
494
|
|
|
495 start_codons = ["ATG","GTG","CTG"]
|
|
|
496 stop_codons = ["TAG","TAA","TGA"]
|
|
|
497
|
|
|
498
|
|
|
499 # Keep track of the longest transcript for each noncoding gene, append this to transcript list later
|
|
|
500 longest_noncoding = {}
|
|
|
501
|
|
|
502
|
|
|
503 tran_count = 0
|
|
|
504 # 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
|
|
|
505 # in the transcript list)
|
|
|
506 genomic_cds_dict = {}
|
|
|
507 tree_dict = {}
|
|
|
508 for transcript in master_dict:
|
|
|
509 #print (transcript, master_dict[transcript]["tran_type"])
|
|
|
510 tran_count += 1
|
|
|
511 if "seq" not in master_dict[transcript]:
|
|
|
512 continue
|
|
|
513 chrom = master_dict[transcript]["chrom"]
|
|
|
514 if chrom not in genomic_cds_dict:
|
|
|
515 genomic_cds_dict[chrom] = []
|
|
|
516 if "cds_start" in master_dict[transcript]:
|
|
|
517 cds_start = master_dict[transcript]["cds_start"]
|
|
|
518 cds_stop = master_dict[transcript]["cds_stop"]
|
|
|
519 if cds_start != "NULL":
|
|
|
520 cds_pos = []
|
|
|
521 for i in range(cds_start, cds_stop+1):
|
|
|
522 cds_pos.append(i)
|
|
|
523
|
|
|
524 for tup in master_dict[transcript]["cds"]:
|
|
|
525 if tup[0] != tup[1]:
|
|
|
526 if tup not in genomic_cds_dict[chrom]:
|
|
|
527 genomic_cds_dict[chrom].append(tup)
|
|
|
528
|
|
|
529 print ("genomic cds dict built")
|
|
|
530 print (list(genomic_cds_dict))
|
|
|
531 for chrom in genomic_cds_dict:
|
|
|
532 tree_dict[chrom] = IntervalTree.from_tuples(genomic_cds_dict[chrom])
|
|
|
533
|
|
|
534 #print (list(tree_dict))
|
|
|
535
|
|
|
536
|
|
|
537 connection = sqlite3.connect("{}".format(sys.argv[6]))
|
|
|
538 cursor = connection.cursor()
|
|
|
539 cursor.execute("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));")
|
|
|
540 cursor.execute("CREATE TABLE IF NOT EXISTS coding_regions (transcript VARCHAR(50), coding_start INT(6), coding_stop INT(6));")
|
|
|
541 cursor.execute("CREATE TABLE IF NOT EXISTS exons (transcript VARCHAR(50), exon_start INT(6), exon_stop INT(6));")
|
|
|
542 cursor.execute("CREATE TABLE IF NOT EXISTS uorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
543 cursor.execute("CREATE TABLE IF NOT EXISTS ouorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
544 cursor.execute("CREATE TABLE IF NOT EXISTS cds (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
545 cursor.execute("CREATE TABLE IF NOT EXISTS nested (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
546 cursor.execute("CREATE TABLE IF NOT EXISTS odorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
547 cursor.execute("CREATE TABLE IF NOT EXISTS dorf (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
548 cursor.execute("CREATE TABLE IF NOT EXISTS minusone(transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
549 cursor.execute("CREATE TABLE IF NOT EXISTS readthrough (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
550 cursor.execute("CREATE TABLE IF NOT EXISTS plusone (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
551 cursor.execute("CREATE TABLE IF NOT EXISTS noncoding (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
552 cursor.execute("CREATE TABLE IF NOT EXISTS extension (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
553 cursor.execute("CREATE TABLE IF NOT EXISTS inframe_stop (transcript VARCHAR(300), start_codon VARCHAR(10), length INT(6), start INT(6), stop INT(6), sequence VARCHAR(50000), cds_coverage FLOAT(20));")
|
|
|
554 connection.commit();
|
|
|
555
|
|
|
556
|
|
|
557 print ("Finding ORFs")
|
|
|
558 transcript_count = 0
|
|
|
559 total_transcripts = len(list(master_dict))
|
|
|
560 for transcript in master_dict:
|
|
|
561 #print ("transcript",transcript)
|
|
|
562 #if transcript != "ENST00000316448":
|
|
|
563 # continue
|
|
|
564 transcript_count += 1
|
|
|
565 if transcript_count%100 == 0:
|
|
|
566 print ("Transcripts complete: {}/{}".format(transcript_count,total_transcripts))
|
|
|
567 if "seq" not in master_dict[transcript]:
|
|
|
568 print ("transcript {} has no sequence".format(transcript))
|
|
|
569 continue
|
|
|
570 seq = master_dict[transcript]["seq"]
|
|
|
571 cds_start = "NULL"
|
|
|
572 cds_stop = "NULL"
|
|
|
573 transcript_len = len(seq)
|
|
|
574 if "cds_start" in master_dict[transcript]:
|
|
|
575 cds_start = master_dict[transcript]["cds_start"]
|
|
|
576 cds_stop = master_dict[transcript]["cds_stop"]
|
|
|
577
|
|
|
578 #Find out what regions of this transcript overlap with any other coding regions
|
|
|
579 coding_positions = []
|
|
|
580 if cds_start != "NULL":
|
|
|
581 #If this is a coding transcript don't bother checking the CDS
|
|
|
582 for i in range(cds_start,cds_stop):
|
|
|
583 coding_positions.append(i)
|
|
|
584 #check 5' leader
|
|
|
585 chrom, pos_list = tran_to_genome(transcript, 0, cds_start, master_dict)
|
|
|
586 for i in range(0,cds_start):
|
|
|
587 genomic_pos = pos_list[i]
|
|
|
588 overlap = tree_dict[chrom][genomic_pos]
|
|
|
589 if len(overlap) != 0:
|
|
|
590 coding_positions.append(i)
|
|
|
591 #check 3' trailer
|
|
|
592 chrom, pos_list = tran_to_genome(transcript, cds_stop, transcript_len, master_dict)
|
|
|
593 for i in range(cds_stop,transcript_len+1):
|
|
|
594 #print ("i",i)
|
|
|
595 genomic_pos = pos_list[i-cds_stop]
|
|
|
596 #print ("genomic position",genomic_pos)
|
|
|
597 overlap = tree_dict[chrom][genomic_pos]
|
|
|
598 if len(overlap) != 0:
|
|
|
599 #print ("overlap not empty appending i",overlap)
|
|
|
600 coding_positions.append(i)
|
|
|
601 else:
|
|
|
602 #check entire transcript
|
|
|
603 chrom, pos_list = tran_to_genome(transcript, 0, transcript_len, master_dict)
|
|
|
604 for i in range(0,transcript_len):
|
|
|
605 genomic_pos = pos_list[i]
|
|
|
606 overlap = tree_dict[chrom][genomic_pos]
|
|
|
607 if len(overlap) != 0:
|
|
|
608 coding_positions.append(i)
|
|
|
609 coding_positions_tuple = to_ranges(coding_positions)
|
|
|
610 coding_dict[transcript] = coding_positions_tuple
|
|
|
611 coding_positions = set(coding_positions)
|
|
|
612 #if this is a coding transcript find the minusone, readhtrough, plusone co-ordinates
|
|
|
613 if cds_start != "NULL":
|
|
|
614 if pseudo_utr_len != 0:
|
|
|
615 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
|
|
|
616 recoding_dict = {0:"minusone",1:"readthrough",2:"plusone"}
|
|
|
617 for addition in recoding_dict:
|
|
|
618 orftype = recoding_dict[addition]
|
|
|
619 for i in range(cds_stop+addition,transcript_len,3):
|
|
|
620 if seq[i:i+3] in stop_codons:
|
|
|
621 orf_seq = seq[cds_stop:i+3]
|
|
|
622 orf_start = cds_stop
|
|
|
623 orf_stop = i+2 # +2 so it refers to the end of the stop codon
|
|
|
624 start_codon = None
|
|
|
625 if orf_seq:
|
|
|
626 length = len(orf_seq)
|
|
|
627 orf_pos_list = []
|
|
|
628 #determine how many nucleotides in this orf overlap with an annotated coding region
|
|
|
629 cds_cov_count = 0.0
|
|
|
630 for position in range(orf_start,orf_stop):
|
|
|
631 orf_pos_list.append(position)
|
|
|
632 for pos in range(orf_start, orf_stop+1):
|
|
|
633 if pos in coding_positions:
|
|
|
634 cds_cov_count += 1
|
|
|
635 cds_cov = cds_cov_count/length
|
|
|
636 cursor.execute("INSERT INTO {} VALUES('{}','{}',{},{},{},'{}',{});".format(orftype, transcript, start_codon, length,orf_start,orf_stop,orf_seq,cds_cov))
|
|
|
637 break
|
|
|
638 for frame in [0,1,2]:
|
|
|
639 for i in range(frame,transcript_len,3):
|
|
|
640 if seq[i:i+3] in start_codons:
|
|
|
641 for x in range(i, transcript_len,3):
|
|
|
642 if seq[x:x+3] in stop_codons:
|
|
|
643 orf_seq = seq[i:x+3]
|
|
|
644 orf_start = i
|
|
|
645 orf_stop = x+2 # +2 so it refers to the end of the stop codon
|
|
|
646 start_codon = seq[i:i+3]
|
|
|
647 length = len(orf_seq)
|
|
|
648 orf_pos_list = []
|
|
|
649 #determine how many nucleotides in this orf overlap with an annotated coding region
|
|
|
650 cds_cov_count = 0.0
|
|
|
651 for pos in range(orf_start, orf_stop+1):
|
|
|
652 if pos in coding_positions:
|
|
|
653 cds_cov_count += 1
|
|
|
654 cds_cov = float(cds_cov_count)/float(length)
|
|
|
655 # Now determine orf type
|
|
|
656 if cds_start == "NULL":
|
|
|
657 orftype = "noncoding"
|
|
|
658 else:
|
|
|
659 #print ("cds start is not null :{}:".format(cds_start))
|
|
|
660 if orf_start == cds_start and orf_stop == cds_stop:
|
|
|
661 orftype = "cds"
|
|
|
662 elif orf_start < cds_start and orf_stop == cds_stop:
|
|
|
663 orftype = "extension"
|
|
|
664 #special case for extensions, we only take from the orf_start to the cds_start, and re-calculate cds coverage
|
|
|
665 orf_stop = cds_start
|
|
|
666 cds_cov_count = 0.0
|
|
|
667 for pos in range(orf_start, orf_stop+1):
|
|
|
668 if pos in coding_positions:
|
|
|
669 cds_cov_count += 1
|
|
|
670 cds_cov = float(cds_cov_count)/float(length)
|
|
|
671 orf_seq = seq[orf_start:cds_start]
|
|
|
672 length = len(orf_seq)
|
|
|
673 elif orf_start < cds_start and orf_stop <= cds_start:
|
|
|
674 orftype = "uorf"
|
|
|
675 elif orf_start < cds_start and orf_stop > cds_start:
|
|
|
676 orftype = "ouorf"
|
|
|
677 elif orf_start >= cds_start and orf_start <= cds_stop and orf_stop <= cds_stop:
|
|
|
678 if orf_stop == cds_stop:
|
|
|
679 break
|
|
|
680 orftype = "nested"
|
|
|
681 elif orf_start >= cds_start and orf_start <= cds_stop and orf_stop > cds_stop:
|
|
|
682 orftype = "odorf"
|
|
|
683 elif orf_start > cds_stop and orf_stop > cds_stop:
|
|
|
684 orftype = "dorf"
|
|
|
685 if orf_stop > cds_start and orf_stop < cds_stop:
|
|
|
686 if (orf_stop+1)%3 == cds_start%3:
|
|
|
687 orftype = "inframe_stop"
|
|
|
688 if transcript not in orf_dict:
|
|
|
689 orf_dict[orftype][transcript] = []
|
|
|
690 cursor.execute("INSERT INTO {} VALUES('{}','{}',{},{},{},'{}',{});".format(orftype, transcript, start_codon, length,orf_start,orf_stop,orf_seq,cds_cov))
|
|
|
691 break
|
|
|
692 # Used to keep track of the codons at cds_start and cds_stop positions,
|
|
|
693 # If there is an issue with the cds co-ordinates the starts and stops counts will
|
|
|
694 # be much lower than the other count, start with 1 to prevent division by 0
|
|
|
695 nuc_dict = {"stops":{"stops":1,"other":0}, "starts":{"starts":1,"other":0}}
|
|
|
696
|
|
|
697 def calcgc(seq):
|
|
|
698 if seq == "":
|
|
|
699 return "NULL"
|
|
|
700 g_count = 0
|
|
|
701 c_count = 0
|
|
|
702 a_count = 0
|
|
|
703 t_count = 0
|
|
|
704 for char in seq:
|
|
|
705 if char == "A":
|
|
|
706 a_count += 1
|
|
|
707 if char == "T":
|
|
|
708 t_count += 1
|
|
|
709 if char == "G":
|
|
|
710 g_count += 1
|
|
|
711 if char == "C":
|
|
|
712 c_count += 1
|
|
|
713 gc = ((g_count+c_count)/float(len(seq)))*100
|
|
|
714 return round(gc,2)
|
|
|
715
|
|
|
716
|
|
|
717
|
|
|
718
|
|
|
719 for transcript in master_dict:
|
|
|
720 #print ("transcripts", transcript)
|
|
|
721 length = master_dict[transcript]["length"]
|
|
|
722 cds_start = master_dict[transcript]["cds_start"]
|
|
|
723 cds_stop = master_dict[transcript]["cds_stop"]
|
|
|
724 seq = master_dict[transcript]["seq"]
|
|
|
725 strand = master_dict[transcript]["strand"]
|
|
|
726 chrom = master_dict[transcript]["chrom"]
|
|
|
727 gene = master_dict[transcript]["gene_name"]
|
|
|
728 gc = calcgc(seq)
|
|
|
729 five_gc = "NULL"
|
|
|
730 cds_gc = "NULL"
|
|
|
731 three_gc = "NULL"
|
|
|
732 if cds_start != "NULL":
|
|
|
733 five_gc = calcgc(seq[:cds_start])
|
|
|
734 cds_gc = calcgc(seq[cds_start:cds_stop])
|
|
|
735 three_gc = calcgc(seq[cds_stop:])
|
|
|
736 # check that the nucleotide cds_start points to is the first of the start codon
|
|
|
737 # take one becase cds_start is 1 based but python indexing is 0 based
|
|
|
738 start_nuc = seq[cds_start-1:cds_start+2]
|
|
|
739 #print ("start nuc",start_nuc)
|
|
|
740 if start_nuc == "ATG":
|
|
|
741 nuc_dict["starts"]["starts"] += 1
|
|
|
742 else:
|
|
|
743 nuc_dict["starts"]["other"] += 1
|
|
|
744 stop_nuc = seq[cds_stop-3:cds_stop]
|
|
|
745 #print ("stop_nuc",stop_nuc)
|
|
|
746 if stop_nuc in ["TAG","TAA","TGA"]:
|
|
|
747 nuc_dict["stops"]["stops"] += 1
|
|
|
748 else:
|
|
|
749 nuc_dict["stops"]["other"] += 1
|
|
|
750 tran_type = master_dict[transcript]["tran_type"]
|
|
|
751 if coding_genes_dict[gene] == True:
|
|
|
752 gene_type = 1
|
|
|
753 else:
|
|
|
754 gene_type = 0
|
|
|
755 #print ("tran type before",tran_type)
|
|
|
756 if tran_type == "coding":
|
|
|
757 tran_type = 1
|
|
|
758 else:
|
|
|
759 tran_type = 0
|
|
|
760 #print ("tran type after",tran_type)
|
|
|
761 start_list = str(master_dict[transcript]["start_list"]).replace(" ","").strip("[]")
|
|
|
762 stop_list = str(master_dict[transcript]["stop_list"]).replace(" ","").strip("[]")
|
|
|
763 exon_junctions = str(master_dict[transcript]["exon_junctions"]).replace(" ","").strip("[]")
|
|
|
764 principal = master_dict[transcript]["principal"]
|
|
|
765 version = master_dict[transcript]["version"]
|
|
|
766 #print (master_dict[transcript])
|
|
|
767 #print (tran_type)
|
|
|
768 #print (gene_type)
|
|
|
769 #print (principal)
|
|
|
770 #print (version)
|
|
|
771 #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))
|
|
|
772 cursor.execute("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,gc,five_gc,cds_gc,three_gc,chrom))
|
|
|
773
|
|
|
774 for tup in master_dict[transcript]["exon"]:
|
|
|
775 cursor.execute("INSERT INTO exons VALUES('{}',{},{});".format(transcript,tup[0],tup[1]))
|
|
|
776 if transcript in coding_dict:
|
|
|
777 for tup in coding_dict[transcript]:
|
|
|
778 cursor.execute("INSERT INTO coding_regions VALUES('{}',{},{});".format(transcript,tup[0],tup[1]))
|
|
|
779
|
|
|
780 connection.commit()
|
|
|
781 connection.close()
|
|
|
782
|
|
|
783 if (nuc_dict["starts"]["other"]/nuc_dict["starts"]["starts"]) > 0.05:
|
|
|
784 print ("Warning: {} transcripts do not have a an AUG at the CDS start position".format(nuc_dict["starts"]["other"]))
|
|
|
785 if (nuc_dict["stops"]["other"]/nuc_dict["stops"]["stops"]) > 0.05:
|
|
|
786 print ("Warning: {} transcripts do not have a an stop codon at the CDS stop position".format(nuc_dict["stops"]["other"]))
|
|
|
787 if len(notinannotation) >0:
|
|
|
788 print ("Warning: {} transcripts were in the fasta file, but not the annotation file, these will be discarded".format(len(notinannotation)))
|
