-
Notifications
You must be signed in to change notification settings - Fork 0
/
demux_recovery.py
executable file
·169 lines (148 loc) · 7.46 KB
/
demux_recovery.py
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
#!/usr/bin/env python3
from Bio import pairwise2
from Bio import SeqIO
from Bio import Seq
import argparse
import gzip
import logging
import os
import sys
import yaml
# MATCH_SCORE = 2
# NON_MATCH_SCORE = 1
# GAP_SCORE = -3
# GAP_EXTENSION_SCORE = -1
# MATCHING_SCORE_FACTOR = 0.85
# I7_ADAPTER = 'GATCGGAAGAGCACACGTCTGAACTCCAGTCAC'
# REQUIRED_MIN_BARCODE_DIST = 2
# REQUIRED_MIN_BARCODE_LENGTH = 3
if __name__ == "__main__":
############################ Parameter ############################
ap = argparse.ArgumentParser()
ap.add_argument('-i', '--input_fastq', required=True,
default=None, help='Input fastq file')
ap.add_argument('-b', '--samples_file', required=True,
default=None, help='Yaml file with barcodes, sample names and adapter')
ap.add_argument('-o', '--output_dir', required=False,
default='.', help='Output directory for the demultiplexed files .')
ap.add_argument('-c', '--config_file', required=False,
default=os.path.join(os.path.dirname(os.path.realpath(sys.argv[0])),
'demux_recovery.yaml'))
ap.add_argument('-l', '--log_level', required=False,
help ='log level, DEBUG or INFO default is INFO',
default='INFO')
args = vars(ap.parse_args())
if args['log_level'] == 'INFO':
log_level = logging.INFO
else:
log_level = logging.DEBUG
logging.basicConfig(level=log_level)
logger = logging.getLogger('log')
if not os.path.isdir(args['output_dir']):
try:
os.makedirs(args['output_dir'])
except OSError:
logger.critical('Could not create directory {}!'.format(args['output_dir']))
sys.exit(1)
if not os.path.isfile(args['input_fastq']):
logger.critical('Input file {} does not exist!'.format(args['input_fastq']))
sys.exit(1)
if not os.path.isfile(args['samples_file']):
logger.critical('Demux setup file {} does not exist!'.format(args['samples_file']))
# Read barcodes and adapter
with open(args['samples_file'], 'r') as stream:
f = yaml.load(stream)
samples = f['samples']
I7_ADAPTER = f['i7_adapter']
# Read config file
with open(args['config_file'], 'r') as stream:
cfg = yaml.load(stream)
# Determine minimal required length of barcodes that is required for demultiplexing
BARCODE_LENGTH = None
for barcode_length in range(cfg['REQUIRED_MIN_BARCODE_LENGTH'], len(samples[0]['i7_barcode'])):
distances = []
for i in range(0, len(samples)):
for j in range(i + 1, len(samples)):
distances.append(
sum([samples[i]['i7_barcode'][k] != samples[j]['i7_barcode'][k]
for k in range(0, min(len(samples[i]['i7_barcode']),
barcode_length
)
)
])
)
min_dist = min(distances)
if min_dist >= cfg['REQUIRED_MIN_BARCODE_DIST']:
BARCODE_LENGTH = barcode_length
break
logger.info('Determined minimal barcode length of {}, with a barcode distance of {}'.format(BARCODE_LENGTH, cfg['REQUIRED_MIN_BARCODE_DIST']))
# Input setup
if args['input_fastq'][-2:] == 'gz':
handle = gzip.open(args['input_fastq'], 'rt')
seqp = SeqIO.parse(handle, 'fastq')
else:
seqp = SeqIO.parse(args['input_fastq'], 'fastq')
# Output setup
for s in samples:
filename = os.path.join(args['output_dir'], '{}_{}.fastq'.format(s['name'], s['i7_barcode']))
s['fastq'] = open(filename, 'wt')
unassigned_samples = {}
unassigned_samples['fastq'] = open(os.path.join(args['output_dir'],
'unassigned.fastq'
), 'wt')
############################ Processing #############################
record_counter = 0
adapter_found_counter = 0
adapter_position_usable_counter = 0
barcode_assigned_counter = 0
for record in seqp:
record_counter += 1
al = pairwise2.align.localms(str(record.seq), I7_ADAPTER, cfg['MATCH_SCORE'], cfg['NON_MATCH_SCORE'], cfg['GAP_SCORE'], cfg['GAP_EXTENSION_SCORE'], one_alignment_only=True)
alignment_score = al[0][2]
start_position = al[0][3]
end_position = al[0][4]
alignment_visualization_sequence = al[0][0]
alignment_visualization_i7_adapter = al[0][1]
if alignment_score >= int(cfg['MATCHING_SCORE_FACTOR'] * cfg['MATCH_SCORE'] * len(I7_ADAPTER)):
adapter_found_counter += 1
logger.debug('Alignment score: {}, start: {}'.format(alignment_score, start_position))
logger.debug(alignment_visualization_sequence)
logger.debug(alignment_visualization_i7_adapter)
# Is there enough sequence left once the adapter is cut off?
if end_position <= len(record.seq) - BARCODE_LENGTH:
adapter_position_usable_counter += 1
# Get the barcode
barcode = str(record.seq[end_position:end_position + BARCODE_LENGTH])
# Put the barcode into the header
record.description = record.description[0:-1] + barcode
# Trim sequence
#_new_record = SeqRecord(Seq(_sequence, generic_dna), id=_record.id, name=_record.name, description=_record.description, annotations=_record.annotations, letter_annotations=_record.letter_annotations)
letter_annotations = record.letter_annotations.copy()
letter_annotations['phred_quality'] = letter_annotations['phred_quality'][0:start_position]
record.letter_annotations = {}
record.seq = Seq.Seq(str(record.seq[0:start_position]))
record.letter_annotations = letter_annotations
# Matches?
matches = [s['i7_barcode'][0:BARCODE_LENGTH] == barcode for s in samples]
try:
barcode_index = matches.index(True)
assert(matches.count(True) <= 1)
fastq_file = samples[barcode_index]['fastq']
barcode_assigned_counter += 1
logger.debug('Found valid barcode {} in position {}'.format(barcode, end_position))
except ValueError:
# to unassigned
fastq_file = unassigned_samples['fastq']
SeqIO.write(record, fastq_file, 'fastq')
if record_counter % 10000 == 0:
logger.info('Sequences processed: {}, with adapters: {} ({}%), demultiplexed: {} ({}%), barcode assigned: {} ({}%)'.format(
record_counter, adapter_found_counter, adapter_found_counter/record_counter * 100,
adapter_position_usable_counter, adapter_position_usable_counter/record_counter * 100,
barcode_assigned_counter, barcode_assigned_counter/record_counter * 100
))
logger.info(
'Sequences processed: {}, with adapters: {} ({}%), demultiplexed: {} ({}%), barcode assigned: {} ({}%)'.format(
record_counter, adapter_found_counter, adapter_found_counter / record_counter * 100,
adapter_position_usable_counter, adapter_position_usable_counter / record_counter * 100,
barcode_assigned_counter, barcode_assigned_counter / record_counter * 100
))