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#!/usr/bin/env python3
"""
Builds a consensus sequence for each set of input sequences
"""
# Info
__author__ = 'Jason Anthony Vander Heiden'
from presto import __version__, __date__, default_consensus_min_count
# Imports
import os
import sys
from argparse import ArgumentParser
from collections import OrderedDict
from textwrap import dedent
# Presto imports
from presto.Defaults import default_delimiter, default_barcode_field, default_out_args, \
default_consensus_min_freq, default_consensus_min_qual
from presto.Commandline import CommonHelpFormatter, checkArgs, getCommonArgParser, parseCommonArgs
from presto.Annotation import flattenAnnotation, mergeAnnotation, getAnnotationValues, \
annotationConsensus
from presto.IO import getFileType, printLog, printWarning, printError
from presto.Sequence import subsetSeqSet, calculateDiversity, \
qualityConsensus, frequencyConsensus, indexSeqSets, \
calculateSetError, deleteSeqPositions, findGapPositions
from presto.Multiprocessing import SeqResult, manageProcesses, feedSeqQueue, \
collectSeqQueue
def processQueue(alive, data_queue, result_queue, cons_func, cons_args={},
min_count=default_consensus_min_count, primer_field=None, primer_freq=None,
max_gap=None, max_error=None, max_diversity=None,
copy_fields=None, copy_actions=None, delimiter=default_delimiter):
"""
Pulls from data queue, performs calculations, and feeds results queue
Arguments:
alive : a multiprocessing.Value boolean controlling whether processing
continues; when False function returns.
data_queue : a multiprocessing.Queue holding data to process.
result_queue : a multiprocessing.Queue to hold processed results.
cons_func : the function to use for consensus generation.
cons_args : a dictionary of optional arguments for the consensus function.
min_count : threshold number of sequences to define a consensus.
primer_field : the annotation field containing primer names;
if None do not annotate with primer names.
primer_freq : the maximum primer frequency that must be meet to build a consensus;
if None do not filter by primer frequency.
max_gap : the maximum frequency of (., -) characters allowed before
deleting a position; if None do not delete positions.
max_error : the minimum error rate to retain a set;
if None do not calculate error rate.
max_diversity : a threshold defining the average pairwise error rate required to retain a read group;
if None do not calculate diversity.
copy_fields : a list of annotations to copy into consensus sequence annotations;
if None no additional annotations will be copied.
copy_actions : the list of actions to take for each copy_fields;
one of ['set', 'majority', 'min', 'max', 'sum'].
delimiter : a tuple of delimiters for (annotations, field/values, value lists).
Returns:
None
"""
try:
# Iterator over data queue until sentinel object reached
while alive.value:
# Get data from queue
if data_queue.empty(): continue
else: data = data_queue.get()
# Exit upon reaching sentinel
if data is None: break
# Define result dictionary for iteration
result = SeqResult(data.id, data.data)
result.log['BARCODE'] = data.id
result.log['SEQCOUNT'] = len(data)
# Define primer annotations and consensus primer if applicable
if primer_field is None:
primer_ann = None
seq_list = data.data
else:
# Calculate consensus primer
primer_ann = OrderedDict()
prcons = annotationConsensus(data.data, primer_field, delimiter=delimiter)
result.log['PRIMER'] = ','.join(prcons['set'])
result.log['PRCOUNT'] = ','.join([str(c) for c in prcons['count']])
result.log['PRCONS'] = prcons['cons']
result.log['PRFREQ'] = prcons['freq']
if primer_freq is None:
# Retain full sequence set if not in primer consensus mode
seq_list = data.data
primer_ann = mergeAnnotation(primer_ann, {'PRIMER':prcons['set']},
delimiter=delimiter)
primer_ann = mergeAnnotation(primer_ann, {'PRCOUNT':prcons['count']},
delimiter=delimiter)
elif prcons['freq'] >= primer_freq:
# Define consensus subset
seq_list = subsetSeqSet(data.data, primer_field, prcons['cons'],
delimiter=delimiter)
primer_ann = mergeAnnotation(primer_ann, {'PRCONS':prcons['cons']},
delimiter=delimiter)
primer_ann = mergeAnnotation(primer_ann, {'PRFREQ':prcons['freq']},
delimiter=delimiter)
else:
# If set fails primer consensus, feed result queue and continue
result_queue.put(result)
continue
# Check count threshold
cons_count = len(seq_list)
result.log['CONSCOUNT'] = cons_count
if cons_count < min_count:
#print(cons_count, min_count)
# If set fails count threshold, feed result queue and continue
result_queue.put(result)
continue
# Update log with input sequences
for i, s in enumerate(seq_list):
result.log['INSEQ%i' % (i + 1)] = str(s.seq)
# If primer and count filters pass, generate consensus sequence
consensus = cons_func(seq_list, **cons_args)
# Delete positions with gap frequency over max_gap and update log with consensus
if max_gap is not None:
gap_positions = set(findGapPositions(seq_list, max_gap))
result.log['CONSENSUS'] = ''.join([' ' if i in gap_positions else x \
for i, x in enumerate(consensus.seq)])
if 'phred_quality' in consensus.letter_annotations:
result.log['QUALITY'] = ''.join([' ' if i in gap_positions else chr(q + 33) \
for i, q in enumerate(consensus.letter_annotations['phred_quality'])])
consensus = deleteSeqPositions(consensus, gap_positions)
else:
gap_positions = None
result.log['CONSENSUS'] = str(consensus.seq)
if 'phred_quality' in consensus.letter_annotations:
result.log['QUALITY'] = ''.join([chr(q + 33) for q in consensus.letter_annotations['phred_quality']])
# Calculate nucleotide diversity
if max_diversity is not None:
diversity = calculateDiversity(seq_list)
result.log['DIVERSITY'] = diversity
# If diversity exceeds threshold, feed result queue and continue
if diversity > max_diversity:
result_queue.put(result)
continue
# Calculate set error against consensus
if max_error is not None:
# Delete positions if required and calculate error
if gap_positions is not None:
seq_check = [deleteSeqPositions(s, gap_positions) for s in seq_list]
else:
seq_check = seq_list
error = calculateSetError(seq_check, consensus)
result.log['ERROR'] = error
# If error exceeds threshold, feed result queue and continue
if error > max_error:
result_queue.put(result)
continue
# TODO: should move this into an improved annotationConsensus function with an action argument
# Parse copy_field annotations and define consensus annotations
if copy_fields is not None and copy_actions is not None:
copy_ann = OrderedDict()
for f, act in zip(copy_fields, copy_actions):
# Numeric operations
if act == 'min':
vals = getAnnotationValues(seq_list, f, delimiter=delimiter)
copy_ann[f] = '%.12g' % min([float(x or 0) for x in vals])
elif act == 'max':
vals = getAnnotationValues(seq_list, f, delimiter=delimiter)
copy_ann[f] = '%.12g' % max([float(x or 0) for x in vals])
elif act == 'sum':
vals = getAnnotationValues(seq_list, f, delimiter=delimiter)
copy_ann[f] = '%.12g' % sum([float(x or 0) for x in vals])
elif act == 'set':
vals = annotationConsensus(seq_list, f, delimiter=delimiter)
copy_ann[f] = vals['set']
copy_ann['%s_COUNT' % f] = vals['count']
elif act == 'majority':
vals = annotationConsensus(seq_list, f, delimiter=delimiter)
copy_ann[f] = vals['cons']
copy_ann['%s_FREQ' % f] = vals['freq']
else:
copy_ann = None
# Define annotation for output sequence
cons_ann = OrderedDict([('ID', data.id),
('CONSCOUNT', cons_count)])
# Merge addition consensus annotations into output sequence annotations
if primer_ann is not None:
cons_ann = mergeAnnotation(cons_ann, primer_ann, delimiter=delimiter)
if copy_ann is not None:
cons_ann = mergeAnnotation(cons_ann, copy_ann, delimiter=delimiter)
# Add output sequence annotations to consensus sequence
consensus.id = consensus.name = flattenAnnotation(cons_ann, delimiter=delimiter)
consensus.description = ''
result.results = consensus
result.valid = True
# Feed results to result queue
result_queue.put(result)
else:
sys.stderr.write('PID %s> Error in sibling process detected. Cleaning up.\n' \
% os.getpid())
return None
except:
alive.value = False
printError('Processing sequence set with ID: %s' % data.id, exit=False)
raise
return None
def buildConsensus(seq_file, barcode_field=default_barcode_field,
min_count=default_consensus_min_count, min_freq=default_consensus_min_freq,
min_qual=default_consensus_min_qual, primer_field=None, primer_freq=None,
max_gap=None, max_error=None, max_diversity=None,
copy_fields=None, copy_actions=None, dependent=False,
out_file=None, out_args=default_out_args, nproc=None, queue_size=None):
"""
Generates consensus sequences
Arguments:
seq_file : the sample sequence file name
barcode_field : the annotation field containing set IDs
min_count : threshold number of sequences to define a consensus
min_freq : the frequency cutoff to assign a base
min_qual : the quality cutoff to assign a base
primer_field : the annotation field containing primer tags;
if None do not annotate with primer tags
primer_freq : the maximum primer tag frequency that must be meet to build a consensus;
if None do not filter by primer frequency
max_gap : the maximum frequency of (., -) characters allowed before
deleting a position; if None do not delete positions
max_error : a threshold defining the maximum allowed error rate to retain a read group;
if None do not calculate error rate
max_diversity : a threshold defining the average pairwise error rate required to retain a read group;
if None do not calculate diversity
dependent : if False treat barcode group sequences as independent data
copy_fields : a list of annotations to copy into consensus sequence annotations;
if None no additional annotations will be copied
copy_actions : the list of actions to take for each copy_fields;
one of ['set', 'majority', 'min', 'max', 'sum']
out_file : output file name. Automatically generated from the input file if None.
out_args : common output argument dictionary from parseCommonArgs.
nproc : the number of processQueue processes;
if None defaults to the number of CPUs
queue_size : maximum size of the argument queue;
if None defaults to 2*nproc
Returns:
list : a list of successful output file names.
"""
# Print parameter info
log = OrderedDict()
log['START'] = 'BuildConsensus'
log['FILE'] = os.path.basename(seq_file)
log['BARCODE_FIELD'] = barcode_field
log['MIN_COUNT'] = min_count
log['MIN_FREQUENCY'] = min_freq
log['MIN_QUALITY'] = min_qual
log['MAX_GAP'] = max_gap
log['PRIMER_FIELD'] = primer_field
log['PRIMER_FREQUENCY'] = primer_freq
log['MAX_ERROR'] = max_error
log['MAX_DIVERSITY'] = max_diversity
log['DEPENDENT'] = dependent
log['COPY_FIELDS'] = ','.join(copy_fields) if copy_fields is not None else None
log['COPY_ACTIONS'] = ','.join(copy_actions) if copy_actions is not None else None
log['NPROC'] = nproc
printLog(log)
# Set consensus building function
in_type = getFileType(seq_file)
if in_type == 'fastq':
cons_func = qualityConsensus
cons_args = {'min_qual': min_qual,
'min_freq': min_freq,
'dependent': dependent}
elif in_type == 'fasta':
cons_func = frequencyConsensus
cons_args = {'min_freq': min_freq}
else:
printError('Input file must be FASTA or FASTQ.')
# Define feeder function and arguments
index_args = {'field': barcode_field, 'delimiter': out_args['delimiter']}
feed_func = feedSeqQueue
feed_args = {'seq_file': seq_file,
'index_func': indexSeqSets,
'index_args': index_args}
# Define worker function and arguments
work_func = processQueue
work_args = {'cons_func': cons_func,
'cons_args': cons_args,
'min_count': min_count,
'primer_field': primer_field,
'primer_freq': primer_freq,
'max_gap': max_gap,
'max_error': max_error,
'max_diversity': max_diversity,
'copy_fields': copy_fields,
'copy_actions': copy_actions,
'delimiter': out_args['delimiter']}
# Define collector function and arguments
collect_func = collectSeqQueue
collect_args = {'seq_file': seq_file,
'label': 'consensus',
'out_file': out_file,
'out_args': out_args,
'index_field': barcode_field}
# Call process manager
result = manageProcesses(feed_func, work_func, collect_func,
feed_args, work_args, collect_args,
nproc, queue_size)
# Print log
result['log']['END'] = 'BuildConsensus'
printLog(result['log'])
return result['out_files']
def getArgParser():
"""
Defines the ArgumentParser
Returns:
argparse.ArgumentParser: argument parser object.
"""
# Define output file names and header fields
fields = dedent(
'''
output files:
consensus-pass
consensus reads.
consensus-fail
raw reads failing consensus filtering criteria.
output annotation fields:
PRIMER
a comma delimited list of unique primer annotations found within the
barcode read group.
PRCOUNT
a comma delimited list of the corresponding counts of unique primer
annotations.
PRCONS
the majority primer within the barcode read group.
PRFREQ
the frequency of the majority primer.
CONSCOUNT
the count of reads within the barcode read group which contributed to
the consensus sequence. This is the total size of the read group,
minus sequence excluded due to user defined filtering criteria.
''')
# Define ArgumentParser
parser = ArgumentParser(description=__doc__, epilog=fields,
parents=[getCommonArgParser(multiproc=True)],
formatter_class=CommonHelpFormatter, add_help=False)
# Consensus arguments
group_cons = parser.add_argument_group('consensus generation arguments')
group_cons.add_argument('-n', action='store', dest='min_count', type=int, default=default_consensus_min_count,
help='The minimum number of sequences needed to define a valid consensus.')
group_cons.add_argument('--bf', action='store', dest='barcode_field', type=str,
default=default_barcode_field,
help='Position of description barcode field to group sequences by.')
group_cons.add_argument('-q', action='store', dest='min_qual', type=int, default=default_consensus_min_qual,
help='''Consensus quality score cut-off under which an ambiguous character is assigned;
does not apply when quality scores are unavailable.''')
group_cons.add_argument('--freq', action='store', dest='min_freq', type=float, default=default_consensus_min_freq,
help='Fraction of character occurrences under which an ambiguous character is assigned.')
group_cons.add_argument('--maxgap', action='store', dest='max_gap', type=float, default=None,
help='''If specified, this defines a cut-off for the frequency of allowed
gap values for each position. Positions exceeding the threshold
are deleted from the consensus. If not defined, positions are always
retained.''')
group_cons.add_argument('--pf', action='store', dest='primer_field', type=str, default=None,
help='Specifies the field name of the primer annotations')
group_cons.add_argument('--prcons', action='store', dest='primer_freq', type=float, default=None,
help='''Specify to define a minimum primer frequency required to assign a consensus primer,
and filter out sequences with minority primers from the consensus building step.''')
group_cons.add_argument('--cf', nargs='+', action='store', dest='copy_fields', type=str, default=None,
help='''Specifies a set of additional annotation fields to copy into
the consensus sequence annotations.''')
group_cons.add_argument('--act', nargs='+', action='store', dest='copy_actions', default=None,
choices=['min', 'max', 'sum', 'set', 'majority'],
help='''List of actions to take for each copy field which defines how
each annotation will be combined into a single value. The actions
"min", "max", "sum" perform the corresponding mathematical
operation on numeric annotations. The action "set" combines
annotations into a comma delimited list of unique values and
adds an annotation named <FIELD>_COUNT specifying the count
of each item in the set. The action "majority" assigns the
most frequent annotation to the consensus annotation and adds
an annotation named <FIELD>_FREQ specifying the frequency
of the majority value.''')
group_cons.add_argument('--dep', action='store_true', dest='dependent',
help='Specify to calculate consensus quality with a non-independence assumption')
# Mutually exclusive error arguments
group_error = group_cons.add_mutually_exclusive_group()
group_error.add_argument('--maxdiv', action='store', dest='max_diversity', type=float, default=None,
help='''Specify to calculate the nucleotide diversity of each read
group (average pairwise error rate) and remove groups exceeding
the given diversity threshold. Diversity is calculate for all
positions within the read group, ignoring any character filtering
imposed by the -q, --freq and --maxgap arguments.
Mutually exclusive with --maxerror.''')
group_error.add_argument('--maxerror', action='store', dest='max_error', type=float, default=None,
help='''Specify to calculate the error rate of each read group
(rate of mismatches from consensus) and remove groups exceeding
the given error threshold. The error rate is calculated against
the final consensus sequence, which may include masked positions
due to the -q and --freq arguments and may have deleted
positions due to the --maxgap argument.
Mutually exclusive with --maxdiv.''')
return parser
if __name__ == '__main__':
"""
Parses command line arguments and calls main function
"""
# Parse arguments
parser = getArgParser()
checkArgs(parser)
args = parser.parse_args()
args_dict = parseCommonArgs(args)
# Convert case of fields
if args_dict['barcode_field']: args_dict['barcode_field'] = args_dict['barcode_field'].upper()
if args_dict['primer_field']: args_dict['primer_field'] = args_dict['primer_field'].upper()
# Check prcons argument dependencies
if args.primer_freq and not args.primer_field:
parser.error('You must define a primer field with --pf to use the --prcons option')
# Check copy field and action arguments
if bool(args_dict['copy_fields']) ^ bool(args_dict['copy_actions']) or \
len((args_dict['copy_fields'] or '')) != len((args_dict['copy_actions'] or '')):
parser.error('You must specify exactly one copy action (--act) per copy field (--cf)')
# Call buildConsensus for each sample file
del args_dict['seq_files']
if 'out_files' in args_dict: del args_dict['out_files']
for i, f in enumerate(args.__dict__['seq_files']):
args_dict['seq_file'] = f
args_dict['out_file'] = args.__dict__['out_files'][i] \
if args.__dict__['out_files'] else None
buildConsensus(**args_dict)
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