1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
|
#!/usr/bin/env python3
import os
import argparse
import simulate_reads
import plot
import shutil
import sys
def copyhead(unassigned, amount, outfile):
infile = open(unassigned)
outfile = open(outfile, 'w')
i = 0
while i < amount * 4:
outfile.write(infile.readline())
i += 1
outfile.close()
def simulation(reffile1, reffile2, name1, name2, temppath, real_readfiles, gapsize, amount, chunksize, se):
print('Step 1/5')
if not se:
if not os.path.isfile(os.path.join(temppath, 'simulated_reads_' + name1 + '_and_' + name2 + '_' + str(amount) + '_1.fasta')) and not os.path.isfile(os.path.join(temppath, 'simulated_reads_' + name1 + '_and_' + name2 + '_2_+' + str(amount) + '.fasta')):
simulate_reads.main(reffile1, reffile2, name1, name2, temppath, real_readfiles, se, amount, gapsize, chunksize)
else:
print('Simulated reads of %s and %s exist. Skipping.' % (name1, name2))
else:
if not os.path.isfile(os.path.join(temppath, 'simulated_reads_' + name1 + '_and_' + name2 + '_' + str(amount) + '.fasta')):
simulate_reads.main(reffile1, reffile2, name1, name2, temppath, real_readfiles, se, amount, gapsize, chunksize)
else:
print('Simulated reads of %s and %s exist. Skipping.' % (name1, name2))
if not se:
if not os.path.isfile(os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '_1.fastq')):
copyhead(real_readfiles[0], amount, os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '_1.fastq'))
copyhead(real_readfiles[1], amount, os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '_2.fastq'))
else:
if not os.path.isfile(os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '.fastq')):
copyhead(real_readfiles[0], amount, os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '.fastq'))
def training(kmers, name1, name2, temppath, threads, amount, se, skriptpath):
print('Step 2/5')
print('Calculating transition matrix')
if not se:
simulated11 = os.path.join(temppath, 'simulated_reads_' + name1 + '_' + str(amount) + '_1.fasta')
simulated12 = os.path.join(temppath, 'simulated_reads_' + name1 + '_' + str(amount) + '_2.fasta')
simulated21 = os.path.join(temppath, 'simulated_reads_' + name2 + '_' + str(amount) + '_1.fasta')
simulated22 = os.path.join(temppath, 'simulated_reads_' + name2 + '_' + str(amount) + '_2.fasta')
for k in kmers:
if not os.path.isfile(os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_PE.txt')):
os.system('java -jar ' + os.path.join(skriptpath, 'trainer.jar') + ' -a1 ' + simulated11 + ' -a2 ' + simulated12 + ' -b1 ' + simulated21 + ' -b2 ' + simulated22 + ' -na ' + name1 + ' -nb ' + name2 + ' -o ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_PE.txt') + ' -T -t ' + str(threads) + ' -k ' + str(k))
else:
print('Transition matrix for k = %i exists. Skipping.' % k)
else:
simulated11 = os.path.join(temppath, 'simulated_reads_' + name1 + '_' + str(amount) + '.fasta')
simulated21 = os.path.join(temppath, 'simulated_reads_' + name2 + '_' + str(amount) + '.fasta')
for k in kmers:
if not os.path.isfile(os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_SE.txt')):
os.system('java -jar ' + os.path.join(skriptpath, 'trainer.jar') + ' -a1 ' + simulated11 + ' -b1 ' + simulated21 + ' -na ' + name1 + ' -nb ' + name2 + ' -o ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_SE.txt') + ' -T -t ' + str(threads) + ' -k ' + str(k))
else:
print('Transition matrix for k = %i exists. Skipping.' % k)
def assign_simulated(kmers, temppath, name1, name2, threads, amount, se, skriptpath):
print('Step 3/5')
print('Assigning subset of simulated reads')
if not se:
for k in kmers:
if not os.path.isfile(os.path.join(temppath, 'classified_simulated_reads_k' + str(k) + '_' + str(amount) + '_1.fasta')):
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + os.path.join(temppath, 'simulated_reads_' + name1 + '_and_' + name2 + '_' + str(amount) + '_1.fasta') + ' -2 ' + os.path.join(temppath, 'simulated_reads_' + name1 + '_and_' + name2 + '_' + str(amount) + '_2.fasta') + ' -d ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_PE.txt') + ' -c MMClassifier -n 100 -o1 ' + os.path.join(temppath, 'classified_simulated_reads_k' + str(k) + '_' + str(amount) + '_1.fasta') + ' -o2 ' + os.path.join(temppath, 'classified_simulated_reads_k' + str(k) + '_' + str(amount) + '_2.fasta') + ' -t ' + str(threads))
else:
print('Assigned simulated reads for k = %i exist. Skipping' % k)
else:
for k in kmers:
if not os.path.isfile(os.path.join(temppath, 'classified_simulated_reads_k' + str(k) + '_' + str(amount) + '.fasta')):
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + os.path.join(temppath, 'simulated_reads_' + name1 + '_and_' + name2 + '_' + str(amount) + '.fasta') + ' -d ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_SE.txt') + ' -c MMClassifier -n 100 -o1 ' + os.path.join(temppath, 'classified_simulated_reads_k' + str(k) + '_' + str(amount) + '.fasta') + ' -t 1')
else:
print('Assigned simulated reads for k = %i exist. Skipping' % k)
def assign_real(kmers, temppath, name1, name2, threads, amount, se, skriptpath):
print('Step 4/5')
print('Assigning subset of real reads')
if not se:
for k in kmers:
if not os.path.isfile(os.path.join(temppath, 'classified_real_reads_k' + str(k) + '_' + str(amount) + '_1.fastq')):
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '_1.fastq') + ' -2 ' + os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '_2.fastq') + ' -d ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_PE.txt') + ' -c MMClassifier -n 100 -o1 ' + os.path.join(temppath, 'classified_real_reads_k' + str(k) + '_' + str(amount) + '_1.fastq') + ' -o2 ' + os.path.join(temppath, 'classified_real_reads_k' + str(k) + '_' + str(amount) + '_2.fastq') + ' -t ' + str(threads))
else:
print('Assigned reads for k = %i exist. Skipping.' % k)
else:
for k in kmers:
if not os.path.isfile(os.path.join(temppath, 'classified_real_reads_k' + str(k) + '_' + str(amount) + '.fastq')):
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + os.path.join(temppath, 'unassigned_subsample_' + str(amount) + '.fastq') + ' -d ' + os.path.join(
temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(k) + '_' + str(amount) + '_SE.txt') + ' -c MMClassifier -n 100 -o1 ' + os.path.join(temppath, 'classified_real_reads_k' + str(k) + '_' + str(amount) + '.fastq') + ' -t ' + str(threads))
else:
print('Assigned reads for k = %i exist. Skipping.' % k)
def do_plots(kmers, outpath, temppath, name1, name2, amount, se, filetype):
if not se:
for k in kmers:
if not os.path.isfile(
os.path.join(outpath, 'ROCplot_k' + str(k) + '_' + str(amount) + '_PE.png')) or not os.path.isfile(
os.path.join(outpath, 'score_histogram_k' + str(k) + '_' + str(amount) + '_PE.png')) or not os.path.isfile(
os.path.join(outpath, 'fitted_histograms_k' + str(k) + '_' + str(amount) + '_PE.png')):
plot.main(k, outpath, name1, name2, os.path.join(temppath, 'classified_simulated_reads_k' + str(k) + '_' + str(amount) + '_1.fasta'), os.path.join(temppath, 'classified_real_reads_k' + str(k) + '_' + str(amount) + '_1.fastq'), amount, se, filetype)
else:
print('Plots for k = %i exist. Skipping' % k)
else:
for k in kmers:
if not os.path.isfile(os.path.join(outpath, 'ROCplot_k' + str(k) + '_' + str(amount) + '_SE.png')) or not os.path.isfile(os.path.join(outpath, 'score_histogram_k' + str(k) + '_' + str(amount) + '_SE.png')) or not os.path.isfile(os.path.join(outpath, 'fitted_histograms_k' + str(k) + '_' + str(amount) + '_SE.png')):
plot.main(k, outpath, name1, name2, os.path.join(temppath, 'classified_simulated_reads_k' + str(k) + '_' + str(amount) + '.fasta'), os.path.join(temppath, 'classified_real_reads_k' + str(k) + '_' + str(amount) + '.fastq'), amount, se, filetype)
else:
print('Plots for k = %i exist. Skipping.' % k)
def assign_all(cutoff_higher, cutoff_lower, kmers, amount, unassigned1, unassigned2, se, temppath, outpath, name1, name2, filetype, skriptpath):
if cutoff_higher:
if not se:
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + unassigned1 + ' -2 ' + unassigned2 + ' -c MMClassifier -n 100 -d ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(kmers[0]) + '_' + str(amount) + '_PE.txt') + ' -o1 ' + os.path.join(outpath, name1 + '_cutoff_' + str(cutoff_higher) + '_k_' + str(kmers[0]) + '_1.' + str(filetype)) + ' -o2 ' + os.path.join(outpath, name1 + '_cutoff_' + str(cutoff_higher) + '_k_' + str(kmers[0]) + '_2.' + str(filetype)) + ' -ch ' + str(cutoff_higher))
else:
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + unassigned1 + ' -c MMClassifier -n 100 -d ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(kmers[0]) + '_' + str(amount) + '_SE.txt') + ' -o1 ' + os.path.join(outpath, name1 + '_cutoff_' + str(cutoff_higher) + '_k_' + str(kmers[0]) + '.' + str(filetype)) + ' -ch ' + str(cutoff_higher))
elif cutoff_lower:
if not se:
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + unassigned1 + ' -2 ' + unassigned2 + ' -c MMClassifier -n 100 -d ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(kmers[0]) + '_' + str(amount) + '_PE.txt') + ' -o1 ' + os.path.join(outpath, name2 + '_cutoff_' + str(cutoff_lower) + '_k_' + str(kmers[0]) + '_1.' + str(filetype)) + ' -o2 ' + os.path.join(outpath, name2 + '_cutoff_' + str(cutoff_lower) + '_k_' + str(kmers[0]) + '_2.' + str(filetype)) + ' -cl ' + str(cutoff_lower))
else:
os.system('java -jar ' + os.path.join(skriptpath, 'classifier.jar') + ' -1 ' + unassigned1 + ' -c MMClassifier -n 100 -d ' + os.path.join(temppath, 'transition_matrix_' + name1 + '_' + name2 + '_k' + str(kmers[0]) + '_' + str(amount) + '_SE.txt') + ' -o1 ' + os.path.join(outpath, name2 + '_cutoff_' + str(cutoff_lower) + '_k_' + str(kmers[0]) + '.' + str(filetype)) + ' -cl ' + str(cutoff_lower))
def main():
parser = argparse.ArgumentParser()
parser.add_argument('-r', '--reffile1', help='Reference file of species 1 in fasta-format. Should pair with name1', required=True)
parser.add_argument('-R', '--reffile2', help='Reference file of species 2 in fasta-format. Should pair with name2', required=True)
parser.add_argument('-n', '--name1', help='Name of species 1', default='species1', required=False)
parser.add_argument('-N', '--name2', help='Name of species 2', default='species2', required=False)
parser.add_argument('-1', '--unassigned1', help='Fasta- or fastq-file containing mixed reads.', required=True)
parser.add_argument('-2', '--unassigned2', help='Fasta- or fastq-file containing mixed reads, only required in paired end mode.', default=None, required=False)
parser.add_argument('-k', '--kmersizes', help='Order of Markov-Chain/kmer length. Enter as range (e.g. 4:8) or list (e.g. 4,6,8) or integer (e.g. 8). Default = 8', default='8', required=False)
parser.add_argument('-o', '--outpath', help='Folder to write results to. Default = $name1_$name2/ in your working directory', required=False)
parser.add_argument('-a', '--amount', help='Number of reads to be simulated, default = 50000', default=50000, required=False)
parser.add_argument('-t', '--threads', help='Number of Threads to use', default=1, required=False)
parser.add_argument('-x', '--chunksize', help='Size of chunks created at a time for simulation, default = 100000. Only change if you know what you are doing!', default=100000, required=False)
parser.add_argument('-g', '--gapsize', help='Estimated size of gapsize in case of paired end reads, default = 1', default=1, required=False)
parser.add_argument('-c', '--cutoff_lower', help='Lower cutoff: Output only reads with a score lower than or equal to this value, use m1 for -1')
parser.add_argument('-C', '--cutoff_higher', help='Higher cutoff: Output only reads with a score higher than or equal to this value, use m1 for -1')
parser.add_argument('-d', '--delete_temp', help='\Delete temporary files. Calculations will start from beginning next time.', action='store_true', default=False)
parser.add_argument('-f', '--filetype', help='Type of your input reads. fasta or fastq, default = fastq', default='fastq', required=False)
args = parser.parse_args()
name1 = args.name1
name2 = args.name2
if not args.outpath:
outpath = os.path.join(os.getcwd(), str(name1) + '_' + str(name2))
else:
outpath = args.outpath
outpath = os.path.join(os.getcwd(), outpath)
temppath = os.path.join(outpath, 'temp')
graphicspath = os.path.join(outpath, 'graphics')
skriptpath = os.path.dirname(os.path.realpath(sys.argv[0]))
if not os.path.exists(temppath):
os.makedirs(temppath)
if not os.path.exists(graphicspath):
os.makedirs(graphicspath)
amount = int(args.amount)
kmersarg = args.kmersizes
if ':' in kmersarg:
kmers = range(int(kmersarg.split(':')[0]), int(kmersarg.split(':')[1]) + 1)
elif ',' in kmersarg:
kmers = []
for each in kmersarg.split(','):
kmers.append(int(each))
else:
kmers = [int(kmersarg)]
threads = int(args.threads)
chunksize = int(args.chunksize)
gapsize = int(args.gapsize)
reffile1 = args.reffile1
reffile2 = args.reffile2
unassigned1 = args.unassigned1
unassigned2 = args.unassigned2
cutoff_lower = args.cutoff_lower
cutoff_higher = args.cutoff_higher
filetype = args.filetype
if not unassigned2:
se = True
else:
se = False
# ##Step1: Read Simulation
simulation(reffile1, reffile2, name1, name2, temppath, [unassigned1, unassigned2], gapsize, amount, chunksize, se)
###Step2: Training
training(kmers, name1, name2, temppath, threads, amount, se, skriptpath)
###Step3: Assign simulated reads
assign_simulated(kmers, temppath, name1, name2, 1, amount, se, skriptpath)
###Step4: Assign real reads
assign_real(kmers, temppath, name1, name2, threads, amount, se, skriptpath)
###Step5: Generate Graphics:
do_plots(kmers, graphicspath, temppath, name1, name2, amount, se, filetype)
###Final Step: Assign all reads
if ((cutoff_higher and not cutoff_lower) or (not cutoff_higher and cutoff_lower)) and len(kmers) == 1:
print('Assigning reads. This may take a while.')
assign_all(cutoff_higher, cutoff_lower, kmers, amount, unassigned1, unassigned2, se, temppath, outpath, name1,
name2, filetype, skriptpath)
else:
print('To assign your reads, please choose a cutoff as well as one kmer-size.')
if args.delete_temp:
shutil.rmtree(os.path.join(temppath))
if __name__ == '__main__':
main()
|
