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1 #!/usr/bin/env python
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2 """Looks for the given primer sequences and clips matching SFF reads.
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3
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4 Takes eight command line options, input read filename, input read format,
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5 input primer FASTA filename, type of primers (forward, reverse or reverse-
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6 complement), number of mismatches (currently only 0, 1 and 2 are supported),
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7 minimum length to keep a read (after primer trimming), should primer-less
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8 reads be kept (boolean), and finally the output sequence filename.
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9
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10 Both the primer and read sequences can contain IUPAC ambiguity codes like N.
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11
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12 This supports FASTA, FASTQ and SFF sequence files. Colorspace reads are not
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13 supported.
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14
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15 The mismatch parameter does not consider gapped alignemnts, however the
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16 special case of missing bases at the very start or end of the read is handled.
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17 e.g. a primer sequence CCGACTCGAG will match a read starting CGACTCGAG...
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18 if one or more mismatches are allowed.
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19
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20 This can also be used for stripping off (and optionally filtering on) barcodes.
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21
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22 Note that only the trim/clip values in the SFF file are changed, not the flow
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23 information of the full read sequence.
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24
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25 This script is copyright 2011-2013 by Peter Cock, The James Hutton Institute
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26 (formerly the Scottish Crop Research Institute, SCRI), UK. All rights reserved.
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27 See accompanying text file for licence details (MIT/BSD style).
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28
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29 This is version 0.0.8 of the script. Currently it uses Python's regular
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30 expression engine for finding the primers, which for my needs is fast enough.
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31 """
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32 import sys
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33 import re
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34 from galaxy_utils.sequence.fasta import fastaReader, fastaWriter
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35 from galaxy_utils.sequence.fastq import fastqReader, fastqWriter
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36
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37 if "-v" in sys.argv or "--version" in sys.argv:
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38 print "v0.0.5"
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39 sys.exit(0)
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40
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41 def stop_err(msg, err=1):
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42 sys.stderr.write(msg)
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43 sys.exit(err)
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44
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45 try:
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46 from Bio.Seq import reverse_complement
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47 from Bio.SeqIO.SffIO import SffIterator, SffWriter
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48 except ImportError:
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49 stop_err("Requires Biopython 1.54 or later")
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50 try:
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51 from Bio.SeqIO.SffIO import ReadRocheXmlManifest
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52 except ImportError:
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53 #Prior to Biopython 1.56 this was a private function
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54 from Bio.SeqIO.SffIO import _sff_read_roche_index_xml as ReadRocheXmlManifest
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55
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56 #Parse Command Line
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57 try:
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58 in_file, seq_format, primer_fasta, primer_type, mm, min_len, keep_negatives, out_file = sys.argv[1:]
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59 except ValueError:
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60 stop_err("Expected 8 arguments, got %i:\n%s" % (len(sys.argv)-1, " ".join(sys.argv)))
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61
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62 if in_file == primer_fasta:
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63 stop_err("Same file given as both primer sequences and sequences to clip!")
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64 if in_file == out_file:
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65 stop_err("Same file given as both sequences to clip and output!")
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66 if primer_fasta == out_file:
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67 stop_err("Same file given as both primer sequences and output!")
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68
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69 try:
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70 mm = int(mm)
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71 except ValueError:
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72 stop_err("Expected non-negative integer number of mismatches (e.g. 0 or 1), not %r" % mm)
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73 if mm < 0:
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74 stop_err("Expected non-negtive integer number of mismatches (e.g. 0 or 1), not %r" % mm)
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75 if mm not in [0,1,2]:
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76 raise NotImplementedError
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77
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78 try:
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79 min_len = int(min_len)
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80 except ValueError:
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81 stop_err("Expected non-negative integer min_len (e.g. 0 or 1), not %r" % min_len)
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82 if min_len < 0:
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83 stop_err("Expected non-negtive integer min_len (e.g. 0 or 1), not %r" % min_len)
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84
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85
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86 if keep_negatives.lower() in ["true", "yes", "on"]:
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87 keep_negatives = True
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88 elif keep_negatives.lower() in ["false", "no", "off"]:
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89 keep_negatives = False
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90 else:
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91 stop_err("Expected boolean for keep_negatives (e.g. true or false), not %r" % keep_negatives)
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92
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93
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94 if primer_type.lower() == "forward":
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95 forward = True
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96 rc = False
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97 elif primer_type.lower() == "reverse":
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98 forward = False
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99 rc = False
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100 elif primer_type.lower() == "reverse-complement":
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101 forward = False
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102 rc = True
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103 else:
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104 stop_err("Expected foward, reverse or reverse-complement not %r" % primer_type)
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105
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106
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107 ambiguous_dna_values = {
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108 "A": "A",
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109 "C": "C",
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110 "G": "G",
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111 "T": "T",
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112 "M": "ACM",
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113 "R": "AGR",
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114 "W": "ATW",
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115 "S": "CGS",
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116 "Y": "CTY",
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117 "K": "GTK",
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118 "V": "ACGMRSV",
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119 "H": "ACTMWYH",
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120 "D": "AGTRWKD",
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121 "B": "CGTSYKB",
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122 "X": ".", #faster than [GATCMRWSYKVVHDBXN] or even [GATC]
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123 "N": ".",
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124 }
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125
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126 ambiguous_dna_re = {}
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127 for letter, values in ambiguous_dna_values.iteritems():
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128 if len(values) == 1:
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129 ambiguous_dna_re[letter] = values
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130 else:
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131 ambiguous_dna_re[letter] = "[%s]" % values
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132
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133
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134 def make_reg_ex(seq):
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135 return "".join(ambiguous_dna_re[letter] for letter in seq)
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136
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137 def make_reg_ex_mm(seq, mm):
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138 if mm > 2:
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139 raise NotImplementedError("At most 2 mismatches allowed!")
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140 seq = seq.upper()
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141 yield make_reg_ex(seq)
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142 for i in range(1,mm+1):
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143 #Missing first/last i bases at very start/end of sequence
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144 for reg in make_reg_ex_mm(seq[i:], mm-i):
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145 yield "^" + reg
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146 for reg in make_reg_ex_mm(seq[:-i], mm-i):
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147 yield "$" + reg
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148 if mm >= 1:
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149 for i,letter in enumerate(seq):
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150 #We'll use a set to remove any duplicate patterns
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151 #if letter not in "NX":
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152 pattern = seq[:i] + "N" + seq[i+1:]
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153 assert len(pattern) == len(seq), "Len %s is %i, len %s is %i" \
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154 % (pattern, len(pattern), seq, len(seq))
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155 yield make_reg_ex(pattern)
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156 if mm >=2:
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157 for i,letter in enumerate(seq):
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158 #We'll use a set to remove any duplicate patterns
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159 #if letter not in "NX":
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160 for k,letter in enumerate(seq[i+1:]):
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161 #We'll use a set to remove any duplicate patterns
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162 #if letter not in "NX":
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163 pattern = seq[:i] + "N" + seq[i+1:i+1+k] + "N" + seq[i+k+2:]
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164 assert len(pattern) == len(seq), "Len %s is %i, len %s is %i" \
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165 % (pattern, len(pattern), seq, len(seq))
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166 yield make_reg_ex(pattern)
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167
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168 def load_primers_as_re(primer_fasta, mm, rc=False):
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169 #Read primer file and record all specified sequences
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170 primers = set()
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171 in_handle = open(primer_fasta, "rU")
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172 reader = fastaReader(in_handle)
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173 count = 0
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174 for record in reader:
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175 if rc:
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176 seq = reverse_complement(record.sequence)
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177 else:
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178 seq = record.sequence
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179 #primers.add(re.compile(make_reg_ex(seq)))
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180 count += 1
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181 for pattern in make_reg_ex_mm(seq, mm):
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182 primers.add(pattern)
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183 in_handle.close()
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184 #Use set to avoid duplicates, sort to have longest first
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185 #(so more specific primers found before less specific ones)
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186 primers = sorted(set(primers), key=lambda p: -len(p))
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187 return count, re.compile("|".join(primers)) #make one monster re!
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188
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189
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190
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191 #Read primer file and record all specified sequences
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192 count, primer = load_primers_as_re(primer_fasta, mm, rc)
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193 print "%i primer sequences" % count
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194
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195 short_neg = 0
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196 short_clipped = 0
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197 clipped = 0
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198 negs = 0
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199
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200 if seq_format.lower()=="sff":
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201 #SFF is different because we just change the trim points
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202 if forward:
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203 def process(records):
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204 global short_clipped, short_neg, clipped, negs
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205 for record in records:
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206 left_clip = record.annotations["clip_qual_left"]
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207 right_clip = record.annotations["clip_qual_right"]
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208 seq = str(record.seq)[left_clip:right_clip].upper()
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209 result = primer.search(seq)
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210 if result:
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211 #Forward primer, take everything after it
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212 #so move the left clip along
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213 if len(seq) - result.end() >= min_len:
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214 record.annotations["clip_qual_left"] = left_clip + result.end()
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215 clipped += 1
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216 yield record
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217 else:
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218 short_clipped += 1
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219 elif keep_negatives:
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220 if len(seq) >= min_len:
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221 negs += 1
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222 yield record
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223 else:
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224 short_neg += 1
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225 else:
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226 def process(records):
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227 global short_clipped, short_neg, clipped, negs
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228 for record in records:
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229 left_clip = record.annotations["clip_qual_left"]
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230 right_clip = record.annotations["clip_qual_right"]
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231 seq = str(record.seq)[left_clip:right_clip].upper()
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232 result = primer.search(seq)
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233 if result:
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234 #Reverse primer, take everything before it
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235 #so move the right clip back
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236 new_len = result.start()
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237 if new_len >= min_len:
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238 record.annotations["clip_qual_right"] = left_clip + new_len
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239 clipped += 1
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240 yield record
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241 else:
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242 short_clipped += 1
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243 elif keep_negatives:
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244 if len(seq) >= min_len:
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245 negs += 1
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246 yield record
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247 else:
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248 short_neg += 1
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249
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250 in_handle = open(in_file, "rb")
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251 try:
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252 manifest = ReadRocheXmlManifest(in_handle)
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253 except ValueError:
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254 manifest = None
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255 in_handle.seek(0)
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256 out_handle = open(out_file, "wb")
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257 writer = SffWriter(out_handle, xml=manifest)
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258 writer.write_file(process(SffIterator(in_handle)))
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259 #End of SFF code
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260 elif seq_format.lower().startswith("fastq"):
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261 in_handle = open(in_file, "rU")
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262 out_handle = open(out_file, "w")
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263 reader = fastqReader(in_handle)
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264 writer = fastqWriter(out_handle)
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265 if forward:
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266 for record in reader:
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267 seq = record.sequence.upper()
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268 result = primer.search(seq)
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269 if result:
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270 #Forward primer, take everything after it
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271 cut = result.end()
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272 record.sequence = seq[cut:]
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273 if len(record.sequence) >= min_len:
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274 record.quality = record.quality[cut:]
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275 clipped += 1
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276 writer.write(record)
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277 else:
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278 short_clipped += 1
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279 elif keep_negatives:
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280 if len(record) >= min_len:
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281 negs += 1
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282 writer.write(record)
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283 else:
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284 short_negs += 1
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285 else:
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286 for record in reader:
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287 seq = record.sequence.upper()
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288 result = primer.search(seq)
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289 if result:
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290 #Reverse primer, take everything before it
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291 cut = result.start()
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292 record.sequence = seq[:cut]
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293 if len(record.sequence) >= min_len:
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294 record.quality = record.quality[:cut]
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295 clipped += 1
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296 writer.write(record)
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297 else:
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298 short_clipped += 1
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299 elif keep_negatives:
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300 if len(record) >= min_len:
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301 negs += 1
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302 writer.write(record)
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303 else:
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304 short_negs += 1
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305 elif seq_format.lower()=="fasta":
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306 in_handle = open(in_file, "rU")
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307 out_handle = open(out_file, "w")
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308 reader = fastaReader(in_handle)
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309 writer = fastaWriter(out_handle)
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310 #Following code is identical to that for FASTQ but without editing qualities
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311 if forward:
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312 for record in reader:
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313 seq = record.sequence.upper()
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314 result = primer.search(seq)
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315 if result:
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316 #Forward primer, take everything after it
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317 cut = result.end()
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318 record.sequence = seq[cut:]
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319 if len(record.sequence) >= min_len:
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320 clipped += 1
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321 writer.write(record)
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322 else:
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323 short_clipped += 1
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324 elif keep_negatives:
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325 if len(record) >= min_len:
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326 negs += 1
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327 writer.write(record)
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328 else:
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329 short_negs += 1
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330 else:
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331 for record in reader:
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332 seq = record.sequence.upper()
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333 result = primer.search(seq)
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334 if result:
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335 #Reverse primer, take everything before it
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336 cut = result.start()
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337 record.sequence = seq[:cut]
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338 if len(record.sequence) >= min_len:
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339 clipped += 1
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340 writer.write(record)
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341 else:
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342 short_clipped += 1
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343 elif keep_negatives:
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344 if len(record) >= min_len:
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345 negs += 1
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346 writer.write(record)
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347 else:
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348 short_negs += 1
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349 else:
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350 stop_err("Unsupported file type %r" % seq_format)
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351 in_handle.close()
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352 out_handle.close()
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353
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354 print "Kept %i clipped reads," % clipped
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355 print "discarded %i short." % short_clipped
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356 if keep_negatives:
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357 print "Kept %i non-matching reads," % negs
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358 print "discarded %i short." % short_neg
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