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#! /usr/bin/env python
# This file is part of khmer, https://github.com/dib-lab/khmer/, and is
# Copyright (C) 2011-2015, Michigan State University.
# Copyright (C) 2015-2016, The Regents of the University of California.
#
# Redistribution and use in source and binary forms, with or without
# modification, are permitted provided that the following conditions are
# met:
#
# * Redistributions of source code must retain the above copyright
# notice, this list of conditions and the following disclaimer.
#
# * Redistributions in binary form must reproduce the above
# copyright notice, this list of conditions and the following
# disclaimer in the documentation and/or other materials provided
# with the distribution.
#
# * Neither the name of the Michigan State University nor the names
# of its contributors may be used to endorse or promote products
# derived from this software without specific prior written
# permission.
#
# THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS
# "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT
# LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR
# A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT
# HOLDER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL,
# SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT
# LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE,
# DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY
# THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT
# (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE
# OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
#
# Contact: khmer-project@idyll.org
# pylint: disable=missing-docstring
"""
Eliminate surplus reads.
Eliminate reads with median k-mer abundance higher than
DESIRED_COVERAGE. Output sequences will be placed in 'infile.keep', with the
option to output to STDOUT.
% python scripts/normalize-by-median.py [ -C <cutoff> ] <data1> <data2> ...
Use '-h' for parameter help.
"""
from contextlib import nullcontext
import sys
import screed
import os
import khmer
import textwrap
from khmer import khmer_args, Countgraph
from contextlib import contextmanager
from khmer.khmer_args import (build_counting_args, add_loadgraph_args,
report_on_config, calculate_graphsize,
sanitize_help, check_argument_range)
from khmer.khmer_args import FileType as khFileType
import argparse
from khmer.kfile import (check_space, check_space_for_graph,
check_valid_file_exists, add_output_compression_type,
FileWriter, describe_file_handle)
from khmer.utils import (write_record, broken_paired_reader, ReadBundle,
clean_input_reads)
from khmer.khmer_logger import (configure_logging, log_info, log_error)
DEFAULT_DESIRED_COVERAGE = 20
class WithDiagnostics(object):
"""
Generator/context manager to do boilerplate output of statistics.
uses a Normalizer object.
"""
def __init__(self, norm, report_fp=None, report_frequency=100000):
self.norm = norm
self.report_fp = report_fp
if report_fp:
report_fp.write('total,kept,f_kept\n')
self.total = 0
self.kept = 0
self.report_frequency = report_frequency
self.next_report_at = self.report_frequency
self.last_report_at = self.report_frequency
def __call__(self, reader, ifilename):
norm = self.norm
report_fp = self.report_fp
reads_start = self.total
total = self.total
kept = self.kept
try:
for _, is_paired, read0, read1 in reader:
if is_paired:
total += 2
else:
total += 1
# do diginorm
for record in norm(is_paired, read0, read1):
kept += 1
yield record
# report!
if total >= self.next_report_at:
self.next_report_at += self.report_frequency
self.last_report_at = total
perc_kept = kept / float(total)
log_info('... kept {kept} of {tot} or {perc_kept:.1%} so'
'far', kept=kept, tot=total,
perc_kept=perc_kept)
log_info('... in file {name}', name=ifilename)
if report_fp:
print("{total},{kept},{f_kept:.4}"
.format(total=total, f_kept=perc_kept,
kept=kept),
file=report_fp)
report_fp.flush()
finally:
self.total = total
self.kept = kept
# per file diagnostic output
if total == reads_start:
log_info('SKIPPED empty file {name}', name=ifilename)
else:
perc_kept = kept / float(total)
log_info('DONE with {inp}; kept {kept} of {total} or '
'{perc_kept:.1%}', inp=ifilename, kept=kept, total=total,
perc_kept=perc_kept)
# make sure there's at least one report per file, at the end of each
# file.
if report_fp and total != self.last_report_at:
perc_kept = kept / float(total)
print("{total},{kept},{f_kept:.4}"
.format(total=total, f_kept=perc_kept, kept=kept),
file=report_fp)
report_fp.flush()
class Normalizer(object):
"""Digital normalization algorithm."""
def __init__(self, desired_coverage, countgraph):
self.countgraph = countgraph
self.desired_coverage = desired_coverage
def __call__(self, is_paired, read0, read1):
"""
Actually does digital normalization - the core algorithm.
* get one (unpaired) or two (paired) reads;
* sanitize the sequences (convert Ns to As);
* get the median k-mer count of one/both reads;
* if any read's median k-mer count is below desired coverage, keep all;
* consume and yield kept reads.
"""
batch = ReadBundle(read0, read1)
desired_coverage = self.desired_coverage
# if any in batch have coverage below desired coverage, consume &yield
if not batch.coverages_at_least(self.countgraph, desired_coverage):
for record in batch.reads:
self.countgraph.consume(record.cleaned_seq)
yield record
@contextmanager
def catch_io_errors(ifile, out, single_out, force, corrupt_files):
"""Context manager to do boilerplate handling of IOErrors."""
try:
yield
except (IOError, OSError, ValueError) as error:
log_error('** ERROR: {error}', error=str(error))
log_error('** Failed on {name}: ', name=ifile)
if not single_out:
os.remove(out.name)
if not force:
log_error('** Exiting!')
sys.exit(1)
else:
log_error('*** Skipping error file, moving on...')
corrupt_files.append(ifile)
def get_parser():
epilog = """\
Discard sequences based on whether or not their median k-mer abundance lies
above a specified cutoff. Kept sequences will be placed in <fileN>.keep.
By default, paired end reads will be considered together; if
either read should be kept, both will be kept. (This keeps both
reads from a fragment, and helps with retention of repeats.)
Unpaired reads are treated individually.
If :option:`-p`/:option:`--paired` is set, then proper pairing is required
and the script will exit on unpaired reads, although
:option:`--unpaired-reads` can be used to supply a file of orphan
reads to be read after the paired reads.
:option:`--force_single` will ignore all pairing information and treat
reads individually.
With :option:`-s`/:option:`--savegraph`, the k-mer countgraph
will be saved to the specified file after all sequences have been
processed. :option:`-l`/:option:`--loadgraph` will load the
specified k-mer countgraph before processing the specified
files. Note that these graphs are are in the same format as those
produced by :program:`load-into-counting.py` and consumed by
:program:`abundance-dist.py`.
To append reads to an output file (rather than overwriting it), send output
to STDOUT with `--output -` and use UNIX file redirection syntax (`>>`) to
append to the file.
Example::
normalize-by-median.py -k 17 tests/test-data/test-abund-read-2.fa
Example::
normalize-by-median.py -p -k 17 \\
tests/test-data/test-abund-read-paired.fa
Example::
normalize-by-median.py -p -k 17 -o - tests/test-data/paired.fq \\
>> appended-output.fq
Example::
normalize-by-median.py -k 17 -f tests/test-data/test-error-reads.fq \\
tests/test-data/test-fastq-reads.fq
Example::
normalize-by-median.py -k 17 -s test.ct \\
tests/test-data/test-abund-read-2.fa \\
tests/test-data/test-fastq-reads.fq"""
parser = build_counting_args(
descr="Do digital normalization (remove mostly redundant sequences)",
epilog=textwrap.dedent(epilog),
citations=['diginorm'])
parser.add_argument('-q', '--quiet', dest='quiet', default=False,
action='store_true')
parser.add_argument('-C', '--cutoff', help="when the median "
"k-mer coverage level is above this number the "
"read is not kept.",
type=check_argument_range(0, 256, "cutoff"),
default=DEFAULT_DESIRED_COVERAGE)
parser.add_argument('-p', '--paired', action='store_true',
help='require that all sequences be properly paired')
parser.add_argument('--force_single', dest='force_single',
action='store_true',
help='treat all sequences as single-ended/unpaired')
parser.add_argument('-u', '--unpaired-reads',
metavar="unpaired_reads_filename",
help='include a file of unpaired reads to which '
'-p/--paired does not apply.')
parser.add_argument('-s', '--savegraph', metavar="filename", default=None,
help='save the k-mer countgraph to disk after all '
'reads are loaded.')
parser.add_argument('-R', '--report',
help='write progress report to report_filename',
metavar='report_filename', type=argparse.FileType('w'))
parser.add_argument('--report-frequency',
metavar='report_frequency', type=int, default=100000,
help='report progress every report_frequency reads')
parser.add_argument('-f', '--force', dest='force',
help='continue past file reading errors',
action='store_true')
parser.add_argument('-o', '--output', metavar="filename",
type=khFileType('wb'),
default=None, dest='single_output_file',
help='only output a single file with '
'the specified filename; use a single dash "-" to '
'specify that output should go to STDOUT (the '
'terminal)')
parser.add_argument('input_filenames', metavar='input_sequence_filename',
help='Input FAST[AQ] sequence filename.', nargs='+')
add_loadgraph_args(parser)
add_output_compression_type(parser)
return parser
def main(): # pylint: disable=too-many-branches,too-many-statements
parser = sanitize_help(get_parser())
args = parser.parse_args()
configure_logging(args.quiet)
report_on_config(args)
report_fp = args.report
force_single = args.force_single
# check for similar filenames
# if we're using a single output file only check for identical filenames
# otherwise, check for identical BASE names as well.
filenames = []
basenames = []
for pathfilename in args.input_filenames:
filenames.append(pathfilename)
if args.single_output_file:
continue # nothing more to worry about
basename = os.path.basename(pathfilename)
if basename in basenames:
log_error('ERROR: Duplicate filename--Cannot handle this!')
log_error('** Exiting!')
sys.exit(1)
basenames.append(basename)
# check that files exist and there is sufficient output disk space.
check_valid_file_exists(args.input_filenames)
check_space(args.input_filenames, args.force)
if args.savegraph is not None:
graphsize = calculate_graphsize(args, 'countgraph')
check_space_for_graph(args.savegraph, graphsize, args.force)
# load or create counting table.
if args.loadgraph:
log_info('loading k-mer countgraph from {graph}',
graph=args.loadgraph)
countgraph = Countgraph.load(args.loadgraph)
else:
log_info('making countgraph')
countgraph = khmer_args.create_countgraph(args)
# create an object to handle diginorm of all files
norm = Normalizer(args.cutoff, countgraph)
with_diagnostics = WithDiagnostics(norm, report_fp, args.report_frequency)
# make a list of all filenames and if they're paired or not;
# if we don't know if they're paired, default to allowing but not
# forcing pairing.
files = []
for element in filenames:
files.append([element, args.paired])
if args.unpaired_reads:
files.append([args.unpaired_reads, False])
corrupt_files = []
outfp = None
output_name = None
if args.single_output_file:
out_single_ctx = FileWriter(args.single_output_file, args.gzip, args.bzip)
else:
out_single_ctx = nullcontext()
if '-' in filenames or '/dev/stdin' in filenames:
print("Accepting input from stdin; output filename must "
"be provided with '-o'.", file=sys.stderr)
sys.exit(1)
with out_single_ctx as out_single_fp:
#
# main loop: iterate over all files given, do diginorm.
#
for filename, require_paired in files:
if not args.single_output_file:
output_name = os.path.basename(filename) + '.keep'
out_ctx = FileWriter(open(output_name, 'wb'), args.gzip,
args.bzip, steal_ownership=True)
else:
out_ctx = nullcontext(enter_result=out_single_fp)
with out_ctx as outfp:
# failsafe context manager in case an input file breaks
with catch_io_errors(filename, outfp, args.single_output_file,
args.force, corrupt_files):
screed_iter = clean_input_reads(screed.open(filename))
reader = broken_paired_reader(screed_iter,
min_length=args.ksize,
force_single=force_single,
require_paired=require_paired)
# actually do diginorm
for record in with_diagnostics(reader, filename):
if record is not None:
write_record(record, outfp)
log_info('output in {name}',
name=describe_file_handle(outfp))
# finished - print out some diagnostics.
log_info('Total number of unique k-mers: {umers}',
umers=countgraph.n_unique_kmers())
if args.savegraph is not None:
log_info('...saving to {name}', name=args.savegraph)
countgraph.save(args.savegraph)
fp_rate = \
khmer.calc_expected_collisions(countgraph, False, max_false_pos=.8)
# for max_false_pos see Zhang et al., http://arxiv.org/abs/1309.2975
log_info('fp rate estimated to be {fpr:1.3f}', fpr=fp_rate)
if args.force and len(corrupt_files) > 0:
log_error("** WARNING: Finished with errors!")
log_error("** I/O Errors occurred in the following files:")
log_error("\t" + " ".join(corrupt_files))
if __name__ == '__main__':
main()
# vim: set filetype=python tabstop=4 softtabstop=4 shiftwidth=4 expandtab:
# vim: set textwidth=79:
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