def test_cat_sff_files(self):
"cat_sff_files cats sff_files" ""
expected_bases = "tcagGCTAACTGTAACCCTCTTGGCACCCACTAAACGCCAATCTTGCTGGAG" +\
"TGTTTACCAGGCACCCAGCAATGTGAATAGTCActgagcgggctggcaaggc"
# works with no file
obs_flows, obs_header = cat_sff_files([])
self.assertEqual(len(obs_flows), 0)
self.assertEqual(obs_header, None)
# works with one file
obs_flows, obs_header = cat_sff_files([sff_file])
obs_flows = list(obs_flows)
self.assertEqual(obs_header['Magic Number'], "0x2E736666")
self.assertEqual(obs_flows[0].Bases, expected_bases)
self.assertEqual(len(obs_flows), 2)
# works with two files
obs_flows, obs_header = (cat_sff_files([sff_file, sff_file]))
obs_flows = list(obs_flows)
self.assertEqual(obs_header['Magic Number'], "0x2E736666")
self.assertEqual(obs_flows[0].Bases, expected_bases)
self.assertEqual(obs_flows[2].Bases, expected_bases)
self.assertEqual(len(obs_flows), 4)
def test_cat_sff_files(self):
"cat_sff_files cats sff_files"""
expected_bases = "tcagGCTAACTGTAACCCTCTTGGCACCCACTAAACGCCAATCTTGCTGGAG"+\
"TGTTTACCAGGCACCCAGCAATGTGAATAGTCActgagcgggctggcaaggc"
#works with no file
obs_flows, obs_header = cat_sff_files([])
self.assertEqual(len(obs_flows),0)
self.assertEqual(obs_header,None)
#works with one file
obs_flows, obs_header = cat_sff_files([sff_file])
obs_flows = list(obs_flows)
self.assertEqual(obs_header['Magic Number'], "0x2E736666")
self.assertEqual(obs_flows[0].Bases, expected_bases)
self.assertEqual(len(obs_flows),2)
#works with two files
obs_flows, obs_header = (cat_sff_files([sff_file, sff_file]))
obs_flows = list(obs_flows)
self.assertEqual(obs_header['Magic Number'], "0x2E736666")
self.assertEqual(obs_flows[0].Bases, expected_bases)
self.assertEqual(obs_flows[2].Bases, expected_bases)
self.assertEqual(len(obs_flows),4)
def split_sff(sff_file_handles, map_file_handle, outdir="/tmp/"):
"""Splits a sff.txt file on barcode/mapping file."""
try:
(flowgrams, header) = cat_sff_files(sff_file_handles)
except ValueError:
# reading in the binary sff usually shows up as ValueError
raise FileFormatError(
'Wrong flogram file format. Make sure you pass the sff.txt format '
+ 'produced by sffinfo. The binary .sff will not work here.')
(inverse_map,
map_count) = build_inverse_barcode_map(parse_fasta(map_file_handle))
filenames = []
# we might have many barcodes and reach python open file limit
# therefor we go the slow way and open and close files each time
# First set up all files with the headers only
for barcode_id in map_count.keys():
fh = open(outdir + barcode_id, "w")
write_sff_header(header, fh, map_count[barcode_id])
fh.close()
filenames.append(outdir + barcode_id)
# Then direct each flowgram into its barcode file
for f in flowgrams:
if f.Name in inverse_map:
barcode_id = inverse_map[f.Name]
fh = open(outdir + barcode_id, "a")
fh.write(f.createFlowHeader() + "\n")
return filenames
def split_sff(sff_file_handles, map_file_handle, outdir="/tmp/"):
"""Splits a sff.txt file on barcode/mapping file."""
try:
(flowgrams, header) = cat_sff_files(sff_file_handles)
except ValueError:
#reading in the binary sff usually shows up as ValueError
raise FileFormatError, 'Wrong flogram file format. Make sure you pass the sff.txt format '+\
'produced by sffinfo. The binary .sff will not work here.'
(inverse_map, map_count) = build_inverse_barcode_map(MinimalFastaParser(map_file_handle))
filenames = []
#we might have many barcodes and reach python open file limit
# therefor we go the slow way and open and close files each time
#First set up all files with the headers only
for barcode_id in map_count.keys():
fh = open(outdir+barcode_id, "w")
write_sff_header(header, fh, map_count[barcode_id])
fh.close()
filenames.append(outdir+barcode_id)
#Then direct each flowgram into its barcode file
for f in flowgrams:
if inverse_map.has_key(f.Name):
barcode_id = inverse_map[f.Name]
fh = open(outdir+barcode_id, "a")
fh.write(f.createFlowHeader()+"\n")
return filenames
def main(commandline_args=None):
parser, opts, args = parse_command_line_parameters(**script_info)
if not opts.sff_fp:
parser.error('Required option flowgram file path (-i) not specified')
elif not files_exist(opts.sff_fp):
parser.error('Flowgram file path does not exist:\n %s \n Pass a valid one via -i.'
% opts.sff_fp)
if(opts.checkpoint_fp):
bp_fp = opts.checkpoint_fp
if not exists(bp_fp):
parser.error('Specified checkpoint file does not exist: %s' % bp_fp)
#peek into sff.txt files to make sure they are parseable
#cat_sff_fles is lazy and only reads header
flowgrams, header = cat_sff_files(map(open, opts.sff_fp.split(',')))
if(opts.split and opts.preprocess_fp):
parser.error('Options --split and --preprocess_fp are exclusive')
if(opts.preprocess_fp):
pp_fp = opts.preprocess_fp
if not exists(opts.preprocess_fp):
parser.error('Specified preprocess directory does not exist: %s' % opts.preprocess_fp)
if not files_exist('%s/prefix_mapping.txt,%s/prefix_dereplicated.fasta' %(pp_fp, pp_fp)):
parser.error('Specified preprocess directory does not contain expected files: ' +\
'prefix_mapping.txt and prefix_dereplicated.fasta')
if opts.titanium:
opts.error_profile = DENOISER_DATA_DIR+'Titanium_error_profile.dat'
opts.low_cutoff = 4
opts.high_cutoff = 5
if not exists(opts.error_profile):
parser.error('Specified error profile %s does not exist' % opts.error_profile)
if opts.output_dir:
#make sure it always ends on /
tmpoutdir=opts.output_dir+"/"
else:
#make random dir in current dir
tmpoutdir = get_tmp_filename(tmp_dir="", prefix="denoiser_", suffix="/")
create_dir(tmpoutdir, not opts.force)
log_fp = 'denoiser.log'
if opts.split:
denoise_per_sample(opts.sff_fp, opts.fasta_fp, tmpoutdir, opts.cluster,
opts.num_cpus, opts.squeeze, opts.percent_id, opts.bail,
opts.primer, opts.low_cutoff, opts.high_cutoff, log_fp,
opts.low_memory, opts.verbose, opts.error_profile, opts.max_num_iter,
opts.titanium)
else:
denoise_seqs(opts.sff_fp, opts.fasta_fp, tmpoutdir, opts.preprocess_fp, opts.cluster,
opts.num_cpus, opts.squeeze, opts.percent_id, opts.bail, opts.primer,
opts.low_cutoff, opts.high_cutoff, log_fp, opts.low_memory,
opts.verbose, opts.error_profile, opts.max_num_iter, opts.titanium,
opts.checkpoint_fp)
def main(commandline_args=None):
parser, opts, args = parse_command_line_parameters(**script_info)
if not opts.sff_fp:
parser.error('Required option flowgram file path (-i) not specified')
elif not files_exist(opts.sff_fp):
parser.error('Flowgram file path does not exist:\n %s \n Pass a valid one via -i.'
% opts.sff_fp)
if(opts.checkpoint_fp):
bp_fp = opts.checkpoint_fp
if not exists(bp_fp):
parser.error('Specified checkpoint file does not exist: %s' % bp_fp)
#peek into sff.txt files to make sure they are parseable
#cat_sff_fles is lazy and only reads header
flowgrams, header = cat_sff_files(map(open, opts.sff_fp.split(',')))
if(opts.split and opts.preprocess_fp):
parser.error('Options --split and --preprocess_fp are exclusive')
if(opts.preprocess_fp):
pp_fp = opts.preprocess_fp
if not exists(opts.preprocess_fp):
parser.error('Specified preprocess directory does not exist: %s' % opts.preprocess_fp)
if not files_exist('%s/prefix_mapping.txt,%s/prefix_dereplicated.fasta' %(pp_fp, pp_fp)):
parser.error('Specified preprocess directory does not contain expected files: ' +\
'prefix_mapping.txt and prefix_dereplicated.fasta')
if opts.titanium:
opts.error_profile = DENOISER_DATA_DIR+'Titanium_error_profile.dat'
opts.low_cutoff = 4
opts.high_cutoff = 5
if not exists(opts.error_profile):
parser.error('Specified error profile %s does not exist' % opts.error_profile)
if opts.output_dir:
#make sure it always ends on /
tmpoutdir=opts.output_dir+"/"
else:
#make random dir in current dir
tmpoutdir = get_tmp_filename(tmp_dir="", prefix="denoiser_", suffix="/")
create_dir(tmpoutdir, not opts.force)
log_fp = 'denoiser.log'
if opts.split:
denoise_per_sample(opts.sff_fp, opts.fasta_fp, tmpoutdir, opts.cluster,
opts.num_cpus, opts.squeeze, opts.percent_id, opts.bail,
opts.primer, opts.low_cutoff, opts.high_cutoff, log_fp,
opts.low_memory, opts.verbose, opts.error_profile, opts.max_num_iter,
opts.titanium)
else:
denoise_seqs(opts.sff_fp, opts.fasta_fp, tmpoutdir, opts.preprocess_fp, opts.cluster,
opts.num_cpus, opts.squeeze, opts.percent_id, opts.bail, opts.primer,
opts.low_cutoff, opts.high_cutoff, log_fp, opts.low_memory,
opts.verbose, opts.error_profile, opts.max_num_iter, opts.titanium,
opts.checkpoint_fp)
def preprocess(sff_fps, log_fh, fasta_fp=None, out_fp="/tmp/",
verbose=False, squeeze=False,
primer=STANDARD_BACTERIAL_PRIMER):
"""Quality filtering and truncation of flowgrams, followed by denoiser phase I.
sff_fps: List of paths to flowgram files
log_fh: log messages are written to log_fh if it is set to something else than None
fasta_fp: Path to fasta file, formatted as from split_libraries.py.
This files is used to filter the flowgrams in sff_fps. Only reads in
fasta_fp are pulled from sff_fps.
out_fp: path to output directory
verbose: a binary verbose flag
squeeze: a flag that controls if sequences are squeezed before phase I.
Squeezing means consecutive identical nucs are collapsed to one.
primer: The primer sequences of the amplification process. This seq will be
removed from all reads during the preprocessing
"""
flowgrams, header = cat_sff_files(map(open, sff_fps))
if(fasta_fp):
# remove barcodes and sequences tossed by split_libraries, i.e. not in
# fasta_fp
labels = imap(lambda a_b: a_b[0], MinimalFastaParser(open(fasta_fp)))
barcode_mapping = extract_barcodes_from_mapping(labels)
(trunc_sff_fp, l) = truncate_flowgrams_in_SFF(flowgrams, header,
outdir=out_fp,
barcode_mapping=barcode_mapping,
primer=primer)
if verbose:
log_fh.write(
"Sequences in barcode mapping: %d\n" %
len(barcode_mapping))
log_fh.write("Truncated flowgrams written: %d\n" % l)
else:
# just do a simple clean and truncate
(clean_sff_fp, l) = cleanup_sff(flowgrams, header, outdir=out_fp)
if verbose:
log_fh.write("Cleaned flowgrams written: %d\n" % l)
flowgrams, header = lazy_parse_sff_handle(open(clean_sff_fp))
(trunc_sff_fp, l) = truncate_flowgrams_in_SFF(flowgrams, header,
outdir=out_fp, primer=primer)
if verbose:
log_fh.write("Truncated flowgrams written: %d\n" % l)
remove(clean_sff_fp)
if (l == 0):
raise ValueError("No flowgrams left after preprocesing.\n" +
"Check your primer sequence")
# Phase I - cluster seqs which are exact prefixe
if verbose:
log_fh.write("Filter flowgrams by prefix matching\n")
(flowgrams, header) = lazy_parse_sff_handle(open(trunc_sff_fp))
l, orig_l, mapping =\
prefix_filter_flowgrams(flowgrams, squeeze=squeeze)
averaged_sff_fp, seqs = build_averaged_flowgrams(mapping, trunc_sff_fp,
min_coverage=1,
# averaging produces too good flowgrams
# such that the greedy clustering clusters too much.
# Use the cluster centroid
# instead by using
# min_coverage 1
out_fp=out_fp + "/prefix_dereplicated.sff.txt")
remove(trunc_sff_fp)
if verbose:
log_fh.write("Prefix matching: removed %d out of %d seqs\n"
% (orig_l - l, orig_l))
log_fh.write("Remaining number of sequences: %d\n" % l)
log_fh.write(make_stats(mapping) + "\n")
# print representative sequences and mapping
print_rep_seqs(mapping, seqs, out_fp)
store_mapping(mapping, out_fp, "prefix")
return (averaged_sff_fp, l, mapping, seqs)
def preprocess(sff_fps,
log_fh,
fasta_fp=None,
out_fp="/tmp/",
verbose=False,
squeeze=False,
primer=STANDARD_BACTERIAL_PRIMER):
"""Quality filtering and truncation of flowgrams, followed by denoiser phase I.
sff_fps: List of paths to flowgram files
log_fh: log messages are written to log_fh if it is set to something else than None
fasta_fp: Path to fasta file, formatted as from split_libraries.py.
This files is used to filter the flowgrams in sff_fps. Only reads in
fasta_fp are pulled from sff_fps.
out_fp: path to output directory
verbose: a binary verbose flag
squeeze: a flag that controls if sequences are squeezed before phase I.
Squeezing means consecutive identical nucs are collapsed to one.
primer: The primer sequences of the amplification process. This seq will be
removed from all reads during the preprocessing
"""
flowgrams, header = cat_sff_files(map(open, sff_fps))
if (fasta_fp):
# remove barcodes and sequences tossed by split_libraries, i.e. not in
# fasta_fp
labels = imap(lambda a_b: a_b[0], parse_fasta(open(fasta_fp)))
barcode_mapping = extract_barcodes_from_mapping(labels)
(trunc_sff_fp,
l) = truncate_flowgrams_in_SFF(flowgrams,
header,
outdir=out_fp,
barcode_mapping=barcode_mapping,
primer=primer)
if verbose:
log_fh.write("Sequences in barcode mapping: %d\n" %
len(barcode_mapping))
log_fh.write("Truncated flowgrams written: %d\n" % l)
else:
# just do a simple clean and truncate
(clean_sff_fp, l) = cleanup_sff(flowgrams, header, outdir=out_fp)
if verbose:
log_fh.write("Cleaned flowgrams written: %d\n" % l)
flowgrams, header = lazy_parse_sff_handle(open(clean_sff_fp))
(trunc_sff_fp, l) = truncate_flowgrams_in_SFF(flowgrams,
header,
outdir=out_fp,
primer=primer)
if verbose:
log_fh.write("Truncated flowgrams written: %d\n" % l)
remove(clean_sff_fp)
if (l == 0):
raise ValueError("No flowgrams left after preprocesing.\n" +
"Check your primer sequence")
# Phase I - cluster seqs which are exact prefixe
if verbose:
log_fh.write("Filter flowgrams by prefix matching\n")
(flowgrams, header) = lazy_parse_sff_handle(open(trunc_sff_fp))
l, orig_l, mapping =\
prefix_filter_flowgrams(flowgrams, squeeze=squeeze)
averaged_sff_fp, seqs = build_averaged_flowgrams(
mapping,
trunc_sff_fp,
min_coverage=1,
# averaging produces too good flowgrams
# such that the greedy clustering clusters too much.
# Use the cluster centroid
# instead by using
# min_coverage 1
out_fp=out_fp + "/prefix_dereplicated.sff.txt")
remove(trunc_sff_fp)
if verbose:
log_fh.write("Prefix matching: removed %d out of %d seqs\n" %
(orig_l - l, orig_l))
log_fh.write("Remaining number of sequences: %d\n" % l)
log_fh.write(make_stats(mapping) + "\n")
# print representative sequences and mapping
print_rep_seqs(mapping, seqs, out_fp)
store_mapping(mapping, out_fp, "prefix")
return (averaged_sff_fp, l, mapping, seqs)
