def test_basic(self):
fasta_file = tempfile.NamedTemporaryFile(mode='w')
fasta_file.write(">probe1\n")
fasta_file.write("ATCGATCG\n")
fasta_file.write(">probe2\n")
fasta_file.write("GGGGG\n")
fasta_file.seek(0)
p1 = probe.Probe.from_str('CCCCC')
p2 = probe.Probe.from_str('GGGGG')
p3 = probe.Probe.from_str('ATCGTTTT')
p4 = probe.Probe.from_str('ATCGATCG')
p5 = probe.Probe.from_str('NNNNATCG')
input_probes = [p1, p2, p3, p4, p5]
fasta_filter = ff.FastaFilter(fasta_file.name)
fasta_filter.filter(input_probes)
self.assertEqual(fasta_filter.output_probes, [p4, p2])
fasta_file.close()
def test_skip_reverse_complements(self):
fasta_file = tempfile.NamedTemporaryFile(mode='w')
fasta_file.write(">probe1\n")
fasta_file.write("ATCGATCG\n")
fasta_file.write(">probe2 | reverse complement of probe1\n")
fasta_file.write("CGATCGAT\n")
fasta_file.write(">probe3\n")
fasta_file.write("GGGGG\n")
fasta_file.seek(0)
p1 = probe.Probe.from_str('CCCCC')
p2 = probe.Probe.from_str('GGGGG')
p3 = probe.Probe.from_str('ATCGTTTT')
p4 = probe.Probe.from_str('ATCGATCG')
p5 = probe.Probe.from_str('CGATCGAT')
p6 = probe.Probe.from_str('NNNNATCG')
input_probes = [p1, p2, p3, p4, p5, p6]
fasta_filter = ff.FastaFilter(fasta_file.name,
skip_reverse_complements=True)
fasta_filter.filter(input_probes)
self.assertEqual(fasta_filter.output_probes, [p4, p2])
fasta_file.close()
def main(args):
logger = logging.getLogger(__name__)
# Set NCBI API key
if args.ncbi_api_key:
ncbi_neighbors.ncbi_api_key = args.ncbi_api_key
# Read the genomes from FASTA sequences
genomes_grouped = []
genomes_grouped_names = []
for ds in args.dataset:
if ds.startswith('collection:'):
# Process a collection of datasets
collection_name = ds[len('collection:'):]
try:
collection = importlib.import_module(
'catch.datasets.collections.' + collection_name)
except ImportError:
raise ValueError("Unknown dataset collection %s" %
collection_name)
for name, dataset in collection.import_all():
genomes_grouped += [seq_io.read_dataset_genomes(dataset)]
genomes_grouped_names += [name]
elif ds.startswith('download:'):
# Download a FASTA for an NCBI taxonomic ID
taxid = ds[len('download:'):]
if args.write_taxid_acc:
taxid_fn = os.path.join(args.write_taxid_acc,
str(taxid) + '.txt')
else:
taxid_fn = None
if '-' in taxid:
taxid, segment = taxid.split('-')
else:
segment = None
ds_fasta_tf = ncbi_neighbors.construct_fasta_for_taxid(
taxid, segment=segment, write_to=taxid_fn)
genomes_grouped += [
seq_io.read_genomes_from_fasta(ds_fasta_tf.name)
]
genomes_grouped_names += ['taxid:' + str(taxid)]
ds_fasta_tf.close()
elif os.path.isfile(ds):
# Process a custom fasta file with sequences
genomes_grouped += [seq_io.read_genomes_from_fasta(ds)]
genomes_grouped_names += [os.path.basename(ds)]
else:
# Process an individual dataset
try:
dataset = importlib.import_module('catch.datasets.' + ds)
except ImportError:
raise ValueError("Unknown file or dataset '%s'" % ds)
genomes_grouped += [seq_io.read_dataset_genomes(dataset)]
genomes_grouped_names += [ds]
if (args.limit_target_genomes
and args.limit_target_genomes_randomly_with_replacement):
raise Exception(("Cannot --limit-target-genomes and "
"--limit-target-genomes-randomly-with-replacement at "
"the same time"))
elif args.limit_target_genomes:
genomes_grouped = [
genomes[:args.limit_target_genomes] for genomes in genomes_grouped
]
elif args.limit_target_genomes_randomly_with_replacement:
k = args.limit_target_genomes_randomly_with_replacement
genomes_grouped = [
random.choices(genomes, k=k) for genomes in genomes_grouped
]
# Store the FASTA paths of blacklisted genomes
blacklisted_genomes_fasta = []
if args.blacklist_genomes:
for bg in args.blacklist_genomes:
if os.path.isfile(bg):
# Process a custom fasta file with sequences
blacklisted_genomes_fasta += [bg]
else:
# Process an individual dataset
try:
dataset = importlib.import_module('catch.datasets.' + bg)
except ImportError:
raise ValueError("Unknown file or dataset '%s'" % bg)
for fp in dataset.fasta_paths:
blacklisted_genomes_fasta += [fp]
# Setup and verify parameters related to probe length
if not args.lcf_thres:
args.lcf_thres = args.probe_length
if args.probe_stride > args.probe_length:
logger.warning(("PROBE_STRIDE (%d) is greater than PROBE_LENGTH "
"(%d), which is usually undesirable and may lead "
"to undefined behavior"), args.probe_stride,
args.probe_length)
if args.lcf_thres > args.probe_length:
logger.warning(("LCF_THRES (%d) is greater than PROBE_LENGTH "
"(%d), which is usually undesirable and may lead "
"to undefined behavior"), args.lcf_thres,
args.probe_length)
if args.island_of_exact_match > args.probe_length:
logger.warning(("ISLAND_OF_EXACT_MATCH (%d) is greater than "
"PROBE_LENGTH (%d), which is usually undesirable "
"and may lead to undefined behavior"),
args.island_of_exact_match, args.probe_length)
# Setup and verify parameters related to k-mer length in probe map
if args.kmer_probe_map_k:
# Check that k is sufficiently small
if args.kmer_probe_map_k > args.probe_length:
raise Exception(("KMER_PROBE_MAP_K (%d) exceeds PROBE_LENGTH "
"(%d), which is not permitted") %
(args.kmer_probe_map_k, args.probe_length))
# Use this value for the SetCoverFilter, AdapterFilter, and
# the Analyzer
kmer_probe_map_k_scf = args.kmer_probe_map_k
kmer_probe_map_k_af = args.kmer_probe_map_k
kmer_probe_map_k_analyzer = args.kmer_probe_map_k
else:
if args.probe_length <= 20:
logger.warning(("PROBE_LENGTH (%d) is small; you may want to "
"consider setting --kmer-probe-map-k to be "
"small as well in order to be more sensitive "
"in mapping candidate probes to target sequence"),
args.probe_length)
# Use a default k of 20 for the SetCoverFilter and AdapterFilter,
# and 10 for the Analyzer since we would like to be more sensitive
# (potentially at the cost of slower runtime) for the latter
kmer_probe_map_k_scf = 20
kmer_probe_map_k_af = 20
kmer_probe_map_k_analyzer = 10
# Set the maximum number of processes in multiprocessing pools
if args.max_num_processes:
probe.set_max_num_processes_for_probe_finding_pools(
args.max_num_processes)
cluster.set_max_num_processes_for_creating_distance_matrix(
args.max_num_processes)
# Raise exceptions or warn based on use of adapter arguments
if args.add_adapters:
if not (args.adapter_a or args.adapter_b):
logger.warning(("Adapter sequences will be added, but default "
"sequences will be used; to provide adapter "
"sequences, use --adapter-a and --adapter-b"))
else:
if args.adapter_a or args.adapter_b:
raise Exception(
("Adapter sequences were provided with "
"--adapter-a and --adapter-b, but --add-adapters is required "
"to add adapter sequences onto the ends of probes"))
# Do not allow both --small-seq-skip and --small-seq-min, since they
# have different intentions
if args.small_seq_skip is not None and args.small_seq_min is not None:
raise Exception(("Both --small-seq-skip and --small-seq-min were "
"specified, but both cannot be used together"))
# Check arguments involving clustering
if args.cluster_and_design_separately and args.identify:
raise Exception(
("Cannot use --cluster-and-design-separately with "
"--identify, because clustering collapses genome groupings into "
"one"))
if args.cluster_from_fragments and not args.cluster_and_design_separately:
raise Exception(("Cannot use --cluster-from-fragments without also "
"setting --cluster-and-design-separately"))
# Check for whether a custom hybridization function was provided
if args.custom_hybridization_fn:
custom_cover_range_fn = tuple(args.custom_hybridization_fn)
else:
custom_cover_range_fn = None
if args.custom_hybridization_fn_tolerant:
custom_cover_range_tolerant_fn = tuple(
args.custom_hybridization_fn_tolerant)
else:
custom_cover_range_tolerant_fn = None
# Setup the filters
# The filters we use are, in order:
filters = []
# [Optional]
# Fasta filter (ff) -- leave out candidate probes
if args.filter_from_fasta:
ff = fasta_filter.FastaFilter(args.filter_from_fasta,
skip_reverse_complements=True)
filters += [ff]
# [Optional]
# Poly(A) filter (paf) -- leave out probes with stretches of 'A' or 'T'
if args.filter_polya:
polya_length, polya_mismatches = args.filter_polya
if polya_length > args.probe_length:
logger.warning(("Length of poly(A) stretch to filter (%d) is "
"greater than PROBE_LENGTH (%d), which is usually "
"undesirable"), polya_length, args.probe_length)
if polya_length < 10:
logger.warning(("Length of poly(A) stretch to filter (%d) is "
"short, and may lead to many probes being "
"filtered"), polya_length)
if polya_mismatches > 10:
logger.warning(("Number of mismatches to tolerate when searching "
"for poly(A) stretches (%d) is high, and may "
"lead to many probes being filtered"),
polya_mismatches)
paf = polya_filter.PolyAFilter(polya_length, polya_mismatches)
filters += [paf]
# Duplicate filter (df) -- condense all candidate probes that
# are identical down to one; this is not necessary for
# correctness, as the set cover filter achieves the same task
# implicitly, but it does significantly lower runtime by
# decreasing the input size to the set cover filter
# Near duplicate filter (ndf) -- condense candidate probes that
# are near-duplicates down to one using locality-sensitive
# hashing; like the duplicate filter, this is not necessary
# but can significantly lower runtime and reduce memory usage
# (even more than the duplicate filter)
if (args.filter_with_lsh_hamming is not None
and args.filter_with_lsh_minhash is not None):
raise Exception(("Cannot use both --filter-with-lsh-hamming "
"and --filter-with-lsh-minhash"))
if args.filter_with_lsh_hamming is not None:
if args.filter_with_lsh_hamming > args.mismatches:
logger.warning(
("Setting FILTER_WITH_LSH_HAMMING (%d) to be greater "
"than MISMATCHES (%d) may cause the probes to achieve less "
"than the desired coverage"), args.filter_with_lsh_hamming,
args.mismatches)
ndf = near_duplicate_filter.NearDuplicateFilterWithHammingDistance(
args.filter_with_lsh_hamming, args.probe_length)
filters += [ndf]
elif args.filter_with_lsh_minhash is not None:
ndf = near_duplicate_filter.NearDuplicateFilterWithMinHash(
args.filter_with_lsh_minhash)
filters += [ndf]
else:
df = duplicate_filter.DuplicateFilter()
filters += [df]
# Set cover filter (scf) -- solve the problem by treating it as
# an instance of the set cover problem
scf = set_cover_filter.SetCoverFilter(
mismatches=args.mismatches,
lcf_thres=args.lcf_thres,
island_of_exact_match=args.island_of_exact_match,
mismatches_tolerant=args.mismatches_tolerant,
lcf_thres_tolerant=args.lcf_thres_tolerant,
island_of_exact_match_tolerant=args.island_of_exact_match_tolerant,
custom_cover_range_fn=custom_cover_range_fn,
custom_cover_range_tolerant_fn=custom_cover_range_tolerant_fn,
identify=args.identify,
blacklisted_genomes=blacklisted_genomes_fasta,
coverage=args.coverage,
cover_extension=args.cover_extension,
cover_groupings_separately=args.cover_groupings_separately,
kmer_probe_map_k=kmer_probe_map_k_scf,
kmer_probe_map_use_native_dict=args.
use_native_dict_when_finding_tolerant_coverage)
filters += [scf]
# [Optional]
# Adapter filter (af) -- add adapters to both the 5' and 3' ends
# of each probe
if args.add_adapters:
# Set default adapter sequences, if not provided
if args.adapter_a:
adapter_a = tuple(args.adapter_a)
else:
adapter_a = ('ATACGCCATGCTGGGTCTCC', 'CGTACTTGGGAGTCGGCCAT')
if args.adapter_b:
adapter_b = tuple(args.adapter_b)
else:
adapter_b = ('AGGCCCTGGCTGCTGATATG', 'GACCTTTTGGGACAGCGGTG')
af = adapter_filter.AdapterFilter(adapter_a,
adapter_b,
mismatches=args.mismatches,
lcf_thres=args.lcf_thres,
island_of_exact_match=\
args.island_of_exact_match,
custom_cover_range_fn=\
custom_cover_range_fn,
kmer_probe_map_k=kmer_probe_map_k_af)
filters += [af]
# [Optional]
# N expansion filter (nef) -- expand Ns in probe sequences
# to avoid ambiguity
if args.expand_n is not None:
nef = n_expansion_filter.NExpansionFilter(
limit_n_expansion_randomly=args.expand_n)
filters += [nef]
# [Optional]
# Reverse complement (rc) -- add the reverse complement of each
# probe that remains
if args.add_reverse_complements:
rc = reverse_complement_filter.ReverseComplementFilter()
filters += [rc]
# If requested, don't apply the set cover filter
if args.skip_set_cover:
filter_before_scf = filters[filters.index(scf) - 1]
filters.remove(scf)
# Define parameters for clustering sequences
if args.cluster_and_design_separately:
cluster_threshold = args.cluster_and_design_separately
if args.skip_set_cover:
cluster_merge_after = filter_before_scf
else:
cluster_merge_after = scf
cluster_fragment_length = args.cluster_from_fragments
else:
cluster_threshold = None
cluster_merge_after = None
cluster_fragment_length = None
# Design the probes
pb = probe_designer.ProbeDesigner(
genomes_grouped,
filters,
probe_length=args.probe_length,
probe_stride=args.probe_stride,
allow_small_seqs=args.small_seq_min,
seq_length_to_skip=args.small_seq_skip,
cluster_threshold=cluster_threshold,
cluster_merge_after=cluster_merge_after,
cluster_fragment_length=cluster_fragment_length)
pb.design()
# Write the final probes to the file args.output_probes
seq_io.write_probe_fasta(pb.final_probes, args.output_probes)
if (args.print_analysis or args.write_analysis_to_tsv
or args.write_sliding_window_coverage
or args.write_probe_map_counts_to_tsv):
analyzer = coverage_analysis.Analyzer(
pb.final_probes,
args.mismatches,
args.lcf_thres,
genomes_grouped,
genomes_grouped_names,
island_of_exact_match=args.island_of_exact_match,
custom_cover_range_fn=custom_cover_range_fn,
cover_extension=args.cover_extension,
kmer_probe_map_k=kmer_probe_map_k_analyzer,
rc_too=args.add_reverse_complements)
analyzer.run()
if args.write_analysis_to_tsv:
analyzer.write_data_matrix_as_tsv(args.write_analysis_to_tsv)
if args.write_sliding_window_coverage:
analyzer.write_sliding_window_coverage(
args.write_sliding_window_coverage)
if args.write_probe_map_counts_to_tsv:
analyzer.write_probe_map_counts(args.write_probe_map_counts_to_tsv)
if args.print_analysis:
analyzer.print_analysis()
else:
# Just print the number of probes
print(len(pb.final_probes))