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demultiplex.py
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from __future__ import print_function
import os
import re
import gzip
import itertools
import argparse
import time
import logging
__author__ = 'Martin Aryee'
logger = logging.getLogger('root')
#args = {'out_dir':'/PHShome/ma695/tmp', 'min_reads':10}
#base = '/data/joung/sequencing_bcl/131007_M01326_0075_000000000-A6B33/Data/Intensities/BaseCalls'
#args['read1'] = os.path.join(base, 'Undetermined_S0_L001_R1_001.fastq.gz')
#args['read2'] = os.path.join(base, 'Undetermined_S0_L001_R2_001.fastq.gz')
#args['index1'] = os.path.join(base, 'Undetermined_S0_L001_I1_001.fastq.gz')
#args['index2'] = os.path.join(base, 'Undetermined_S0_L001_I2_001.fastq.gz')
def fq(file):
if re.search('.gz$', file):
fastq = gzip.open(file, 'rb')
else:
fastq = open(file, 'r')
with fastq as f:
while True:
l1 = f.readline()
if not l1:
break
l2 = f.readline()
l3 = f.readline()
l4 = f.readline()
yield [l1, l2, l3, l4]
def get_sample_id(i1, i2, sample_names):
seq1 = i1[1]
seq2 = i2[1]
sample_barcode = seq1[1:8] + seq2[1:8]
if sample_barcode in sample_names:
return sample_names[sample_barcode]
else:
return sample_barcode
def demultiplex(read1, read2, index1, index2, sample_barcodes, out_dir, min_reads=10000):
if not os.path.exists(out_dir):
os.makedirs(out_dir)
if type(sample_barcodes) != dict:
sample_names = {}
if not sample_barcodes==None:
for line in open(sample_barcodes, 'r'):
fields = line.strip().split('\t')
if len(fields)==2:
sampleid, barcode = fields
sample_names[barcode] = sampleid
else:
sample_names = sample_barcodes
outfiles_r1 = {}
outfiles_r2 = {}
outfiles_i1 = {}
outfiles_i2 = {}
total_count = 0
count = {}
buffer_r1 = {}
buffer_r2 = {}
buffer_i1 = {}
buffer_i2 = {}
#it = itertools.izip(fq(args['read1']), fq(args['read2']), fq(args['index1']), fq(args['index2']))
#for r1,r2,i1,i2 in itertools.islice(it, 0, 100):
start = time.time()
for r1,r2,i1,i2 in itertools.izip(fq(read1), fq(read2), fq(index1), fq(index2)):
total_count += 1
if total_count % 1000000 == 0:
logger.info("Processed %d reads in %.1f minutes.", total_count, (time.time()-start)/60)
sample_id = get_sample_id(i1, i2, sample_names)
# Increment read count and create output buffers if this is a new sample barcode
if not count.has_key(sample_id):
count[sample_id] = 0
buffer_r1[sample_id] = []
buffer_r2[sample_id] = []
buffer_i1[sample_id] = []
buffer_i2[sample_id] = []
count[sample_id] += 1
# Write the reads to file or to sample-specific output buffers if we haven't
# reached min_reads
# The buffers are used to avoid writing fastqs for sample barcodes with very few reads.
if count[sample_id] < min_reads:
buffer_r1[sample_id].append(r1)
buffer_r2[sample_id].append(r2)
buffer_i1[sample_id].append(i1)
buffer_i2[sample_id].append(i2)
elif count[sample_id] == min_reads:
outfiles_r1[sample_id] = open(os.path.join(out_dir, '%s.r1.fastq' % sample_id), 'w')
outfiles_r2[sample_id] = open(os.path.join(out_dir, '%s.r2.fastq' % sample_id), 'w')
outfiles_i1[sample_id] = open(os.path.join(out_dir, '%s.i1.fastq' % sample_id), 'w')
outfiles_i2[sample_id] = open(os.path.join(out_dir, '%s.i2.fastq' % sample_id), 'w')
# Spill the buffers to sample-specific fastqs
for record in buffer_r1[sample_id] + r1:
outfiles_r1[sample_id].write(''.join(record))
for record in buffer_r2[sample_id] + r2:
outfiles_r2[sample_id].write(''.join(record))
for record in buffer_i1[sample_id] + i1:
outfiles_i1[sample_id].write(''.join(record))
for record in buffer_i2[sample_id] + i2:
outfiles_i2[sample_id].write(''.join(record))
del buffer_r1[sample_id]
del buffer_r2[sample_id]
del buffer_i1[sample_id]
del buffer_i2[sample_id]
else:
for line in r1:
print (line, file=outfiles_r1[sample_id], end="")
for line in r2:
print (line, file=outfiles_r2[sample_id], end="")
for line in i1:
print (line, file=outfiles_i1[sample_id], end="")
for line in i2:
print (line, file=outfiles_i2[sample_id], end="")
# Write remaining buffered reads to a single fastq.
# (These reads correspond to barcodes that were seen less than min_reads times)
undetermined_r1 = open(os.path.join(out_dir, 'undetermined.r1.fastq'), 'w')
undetermined_r2 = open(os.path.join(out_dir, 'undetermined.r2.fastq'), 'w')
undetermined_i1 = open(os.path.join(out_dir, 'undetermined.i1.fastq'), 'w')
undetermined_i2 = open(os.path.join(out_dir, 'undetermined.i2.fastq'), 'w')
for sample_id in buffer_r1.keys():
for record in buffer_r1[sample_id]:
undetermined_r1.write(''.join(record))
for record in buffer_r2[sample_id]:
undetermined_r2.write(''.join(record))
for record in buffer_i1[sample_id]:
undetermined_i1.write(''.join(record))
for record in buffer_i2[sample_id]:
undetermined_i2.write(''.join(record))
# Close files
for sample_id in outfiles_r1:
outfiles_r1[sample_id].close()
outfiles_r2[sample_id].close()
outfiles_i1[sample_id].close()
outfiles_i2[sample_id].close()
undetermined_r1.close()
undetermined_r2.close()
undetermined_i1.close()
undetermined_i2.close()
num_fastqs = len([v for k,v in count.iteritems() if v>=min_reads])
logger.info('Wrote FASTQs for the %d sample barcodes out of %d with at least %d reads.', num_fastqs, len(count), min_reads)
def main():
parser = argparse.ArgumentParser()
parser.add_argument('--read1', required=True)
parser.add_argument('--read2', required=True)
parser.add_argument('--index1', required=True)
parser.add_argument('--index2', required=True)
parser.add_argument('--min_reads', type=int, default=10000)
parser.add_argument('--sample_barcodes')
parser.add_argument('--out_dir', default='.')
args = vars(parser.parse_args())
demultiplex(args['read1'], args['read2'], args['index1'], args['index2'], args['sample_barcodes'], args['out_dir'], min_reads=args['min_reads'])
if __name__ == '__main__':
main()