class ResolveTypes():
"""Resolve cases where a species has multiple genomes assembled from the type strain."""
def __init__(self, ani_cache_file, cpus, output_dir):
"""Initialization."""
self.ltp_dir = 'rna_ltp_132'
self.ltp_results_file = 'ssu.taxonomy.tsv'
self.LTP_METADATA = namedtuple(
'LTP_METADATA',
'taxonomy taxa species ssu_len evalue bitscore aln_len perc_iden perc_aln'
)
self.ltp_pi_threshold = 99.0
self.ltp_pa_threshold = 90.0
self.ltp_ssu_len_threshold = 900
self.ltp_evalue_threshold = 1e-10
self.output_dir = output_dir
self.logger = logging.getLogger('timestamp')
self.cpus = cpus
self.fastani = FastANI(ani_cache_file, cpus)
self.ani_pickle_dir = os.path.join(self.output_dir, 'ani_pickles')
if not os.path.exists(self.ani_pickle_dir):
os.makedirs(self.ani_pickle_dir)
def _parse_ltp_taxonomy_str(self, ltp_taxonomy_str):
"""Parse taxa and species from LTP taxonomy string."""
if ';type sp.|' in ltp_taxonomy_str:
taxa = ltp_taxonomy_str.split(';type sp.|')[0].split(';')
elif ';|' in ltp_taxonomy_str:
taxa = ltp_taxonomy_str.split(';|')[0].split(';')
elif '|' in ltp_taxonomy_str:
taxa = ltp_taxonomy_str.split('|')[0].split(';')
elif ltp_taxonomy_str[-1] == ';':
taxa = ltp_taxonomy_str[0:-1].split(';')
else:
taxa = ltp_taxonomy_str.split(';')
sp = taxa[-1]
if ' subsp. ' in sp:
sp = ' '.join(sp.split()[0:2])
# validate that terminal taxon appears to be a
# valid binomial species name
if (sp[0].islower() or any(c.isdigit() for c in sp)
or any(c.isupper() for c in sp[1:])):
print(ltp_taxonomy_str, taxa)
assert False
return taxa, 's__' + sp
def parse_ltp_metadata(self, type_gids, cur_genomes):
"""Parse Living Tree Project 16S rRNA metadata."""
metadata = defaultdict(list)
for gid in type_gids:
genome_path = os.path.dirname(
os.path.abspath(cur_genomes[gid].genomic_file))
ltp_file = os.path.join(genome_path, self.ltp_dir,
self.ltp_results_file)
if os.path.exists(ltp_file):
with open(ltp_file) as f:
header = f.readline().strip().split('\t')
taxonomy_index = header.index('taxonomy')
ssu_len_index = header.index('length')
evalue_index = header.index('blast_evalue')
bitscore_index = header.index('blast_bitscore')
aln_len_index = header.index('blast_align_len')
pi_index = header.index('blast_perc_identity')
for line in f:
tokens = line.strip().split('\t')
taxonomy = tokens[taxonomy_index]
ssu_len = int(tokens[ssu_len_index])
evalue = float(tokens[evalue_index])
bitscore = float(tokens[bitscore_index])
aln_len = int(tokens[aln_len_index])
pi = float(tokens[pi_index])
taxa, sp = self._parse_ltp_taxonomy_str(taxonomy)
metadata[gid].append(
self.LTP_METADATA(taxonomy=taxonomy,
taxa=taxa,
species=sp,
ssu_len=ssu_len,
evalue=evalue,
bitscore=bitscore,
aln_len=aln_len,
perc_iden=pi,
perc_aln=aln_len * 100.0 /
ssu_len))
return metadata
def ltp_defined_species(self, ltp_taxonomy_file):
"""Get all species present in the LTP database."""
ltp_species = set()
with open(ltp_taxonomy_file, encoding='utf-8') as f:
for line in f:
tokens = line.strip().split('\t')
taxonomy = tokens[1]
_taxa, sp = self._parse_ltp_taxonomy_str(taxonomy)
ltp_species.add(sp)
return ltp_species
def ltp_species(self, gid, ltp_metadata):
"""Get high confident species assignments."""
sp = set()
for hit in ltp_metadata[gid]:
# check if hit should be trusted
if (hit.perc_iden >= self.ltp_pi_threshold
and hit.perc_aln >= self.ltp_pa_threshold
and hit.ssu_len >= self.ltp_ssu_len_threshold
and hit.evalue < self.ltp_evalue_threshold):
sp.add(hit.species)
return sp
def check_strain_ani(self, gid_anis, untrustworthy_gids):
"""Check if genomes meet strain ANI criteria."""
for gid1, gid2 in combinations(gid_anis, 2):
if gid1 in untrustworthy_gids or gid2 in untrustworthy_gids:
continue
if gid_anis[gid1][gid2] < 99:
return False
return True
def resolve_by_intra_specific_ani(self, gid_anis):
"""Resolve by removing intra-specific genomes with divergent ANI values."""
if len(gid_anis) <= 2:
return False, {}
# consider most divergent genome as untrustworthy
untrustworthy_gids = {}
while True:
# find most divergent genome
min_ani = 100
untrustworthy_gid = None
for gid in gid_anis:
if gid in untrustworthy_gids:
continue
anis = [
ani for cur_gid, ani in gid_anis[gid].items()
if cur_gid not in untrustworthy_gids
]
if np_mean(anis) < min_ani:
min_ani = np_mean(anis)
untrustworthy_gid = gid
untrustworthy_gids[
untrustworthy_gid] = f'{min_ani:.2f}% ANI to other type strain genomes'
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
if all_similar:
return True, untrustworthy_gids
remaining_genomes = len(gid_anis) - len(untrustworthy_gids)
if remaining_genomes <= 2 or len(untrustworthy_gids) >= len(
gid_anis):
return False, {}
def resolve_by_ncbi_types(self, gid_anis, type_gids, cur_genomes):
"""Resolve by consulting NCBI type material metadata."""
untrustworthy_gids = {}
ncbi_type_count = 0
for gid in type_gids:
if not cur_genomes[gid].is_ncbi_type_strain():
untrustworthy_gids[
gid] = 'Not classified as assembled from type material at NCBI'
else:
ncbi_type_count += 1
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
if all_similar and len(untrustworthy_gids) > 0 and ncbi_type_count > 0:
return True, untrustworthy_gids
return False, {}
def resolve_by_ncbi_reps(self, gid_anis, type_gids, cur_genomes):
"""Resovle by considering genomes annotated as representative genomes at NCBI."""
untrustworthy_gids = {}
ncbi_rep_count = 0
for gid in type_gids:
if not cur_genomes[gid].is_ncbi_representative():
untrustworthy_gids[
gid] = 'Excluded in favour of RefSeq representative or reference genome'
else:
ncbi_rep_count += 1
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
if all_similar and ncbi_rep_count >= 1:
return True, untrustworthy_gids
return False, {}
def resolve_gtdb_family(self, gid_anis, ncbi_sp, type_gids, cur_genomes):
"""Resolve by identifying genomes with a conflicting GTDB family assignment."""
genus = 'g__' + generic_name(ncbi_sp)
gtdb_genus_rep = cur_genomes.gtdb_type_species_of_genus(genus)
if not gtdb_genus_rep:
return False, {}
expected_gtdb_family = cur_genomes[gtdb_genus_rep].gtdb_taxa.family
untrustworthy_gids = {}
matched_family = 0
for gid in type_gids:
if cur_genomes[gid].gtdb_taxa.family == expected_gtdb_family:
matched_family += 1
else:
# genome is classified to a different GTDB family than
# expected for this species
untrustworthy_gids[
gid] = f'Conflicting GTDB family assignment of {cur_genomes[gid].gtdb_taxa.family}, expected {expected_gtdb_family}'
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
# conflict is resolved if remaining genomes pass ANI similarity test,
if all_similar and len(untrustworthy_gids) > 0 and matched_family > 0:
return True, untrustworthy_gids
return False, {}
def resolve_gtdb_genus(self, gid_anis, ncbi_sp, type_gids, cur_genomes):
"""Resolve by identifying genomes with a conflicting GTDB genus assignments."""
ncbi_genus = 'g__' + generic_name(ncbi_sp)
untrustworthy_gids = {}
matched_genus = 0
for gid in type_gids:
canonical_gtdb_genus = canonical_taxon(
cur_genomes[gid].gtdb_taxa.genus)
if ncbi_genus == canonical_gtdb_genus:
matched_genus += 1
else:
untrustworthy_gids[
gid] = f'Conflicting GTDB genus assignment of {cur_genomes[gid].gtdb_taxa.genus}, expected {ncbi_genus}'
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
if all_similar and len(untrustworthy_gids) > 0 and matched_genus > 0:
return True, untrustworthy_gids
return False, {}
def resolve_gtdb_species(self, gid_anis, ncbi_sp, type_gids, cur_genomes):
"""Resolve by identifying genomes with a conflicting GTDB species assignments to different type material."""
ncbi_sp_epithet = specific_epithet(ncbi_sp)
untrustworthy_gids = {}
matched_sp_epithet = 0
for gid in type_gids:
if ncbi_sp_epithet == cur_genomes[gid].gtdb_taxa.specific_epithet:
matched_sp_epithet += 1
else:
# check if genome is classified to a GTDB species cluster supported
# by a type strain genome in which case we should consider this
# genome untrustworthy
gtdb_sp = cur_genomes[gid].gtdb_taxa.species
if gtdb_sp != 's__':
gtdb_sp_rid = cur_genomes.gtdb_sp_rep(gtdb_sp)
if cur_genomes[gtdb_sp_rid].is_effective_type_strain():
# genome has been assigned to another species
# defined by a type strain genome
ani, af = self.fastani.symmetric_ani_cached(
gid, gtdb_sp_rid, cur_genomes[gid].genomic_file,
cur_genomes[gtdb_sp_rid].genomic_file)
untrustworthy_gids[
gid] = f'Conflicting GTDB species assignment of {cur_genomes[gid].gtdb_taxa.species} [ANI={ani:.2f}%; AF={af:.2f}%]'
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
# conflict is resolved if remaining genomes pass ANI similarity test,
if all_similar and len(
untrustworthy_gids) > 0 and matched_sp_epithet > 0:
return True, untrustworthy_gids
return False, {}
def resolve_validated_untrustworthy_ncbi_genomes(self, gid_anis, ncbi_sp,
type_gids, ltp_metadata,
ltp_defined_species,
cur_genomes):
"""Resolve by identifying genomes marked as `untrustworthy as type` at NCBI and with conflicting LTP assignments."""
if ncbi_sp not in ltp_defined_species:
return False, {}
untrustworthy_gids = {}
for gid in type_gids:
if 'untrustworthy as type' in cur_genomes[
gid].excluded_from_refseq_note.lower():
ltp_species = self.ltp_species(gid, ltp_metadata)
if ncbi_sp not in ltp_species and len(ltp_species) > 0:
untrustworthy_gids[
gid] = f"Conflicting 16S rRNA hits to LTP database of {' / '.join(set(ltp_species))}"
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
# conflict is resolved if remaining genomes pass ANI similarity test,
if all_similar and len(untrustworthy_gids) > 0:
return True, untrustworthy_gids
return False, {}
def resolve_ltp_conflict(self, gid_anis, ncbi_sp, type_gids, ltp_metadata,
require_conflict_sp):
"""Resolve by considering BLAST hits of 16S rRNA genes to LTP database."""
untrustworthy_gids = {}
genomes_matching_expected_sp = 0
for gid in type_gids:
expected_sp_count = 0
match_unexpected_sp = []
for hit in ltp_metadata[gid]:
# check if hit should be trusted
if (hit.perc_iden >= self.ltp_pi_threshold
and hit.perc_aln >= self.ltp_pa_threshold
and hit.ssu_len >= self.ltp_ssu_len_threshold
and hit.evalue < self.ltp_evalue_threshold):
ltp_sp = hit.species
if ltp_sp == ncbi_sp:
expected_sp_count += 1
else:
match_unexpected_sp.append(ltp_sp)
if expected_sp_count == 0 and len(
match_unexpected_sp) >= require_conflict_sp:
if len(match_unexpected_sp) > 0:
untrustworthy_gids[
gid] = f"Conflicting 16S rRNA hits to LTP database of {' / '.join(set(match_unexpected_sp))}"
else:
untrustworthy_gids[
gid] = "Lack of 16S rRNA hits to LTP database"
elif expected_sp_count > len(match_unexpected_sp):
genomes_matching_expected_sp += 1
all_similar = self.check_strain_ani(gid_anis, untrustworthy_gids)
if all_similar and len(
untrustworthy_gids) > 0 and genomes_matching_expected_sp > 0:
return True, untrustworthy_gids
return False, {}
def parse_untrustworthy_type_ledger(self, untrustworthy_type_ledger):
"""Parse file indicating genomes considered to be untrustworthy as type material."""
manual_untrustworthy_types = {}
with open(untrustworthy_type_ledger) as f:
header = f.readline().strip().split('\t')
ncbi_sp_index = header.index('NCBI species')
reason_index = header.index('Reason for declaring untrustworthy')
for line in f:
tokens = line.strip().split('\t')
gid = canonical_gid(tokens[0])
manual_untrustworthy_types[gid] = (tokens[ncbi_sp_index],
tokens[reason_index])
return manual_untrustworthy_types
def sp_with_mult_type_strains(self, cur_genomes):
"""Identify NCBI species with multiple type strain of species genomes."""
sp_type_strain_genomes = defaultdict(set)
for gid in cur_genomes:
if cur_genomes[gid].is_effective_type_strain():
ncbi_sp = cur_genomes[gid].ncbi_taxa.species
if ncbi_sp != 's__':
# yes, NCBI has genomes marked as assembled from type material
# that do not actually have a binomial species name
sp_type_strain_genomes[ncbi_sp].add(gid)
multi_type_strains_sp = {
ncbi_sp: gids
for ncbi_sp, gids in sp_type_strain_genomes.items()
if len(gids) > 1
}
return multi_type_strains_sp
def calculate_type_strain_ani(self, ncbi_sp, type_gids, cur_genomes,
use_pickled_results):
"""Calculate pairwise ANI between type strain genomes."""
ncbi_sp_str = ncbi_sp[3:].lower().replace(' ', '_')
if not use_pickled_results: # ***
ani_af = self.fastani.pairwise(type_gids,
cur_genomes.genomic_files)
pickle.dump(
ani_af,
open(os.path.join(self.ani_pickle_dir, f'{ncbi_sp_str}.pkl'),
'wb'))
else:
ani_af = pickle.load(
open(os.path.join(self.ani_pickle_dir, f'{ncbi_sp_str}.pkl'),
'rb'))
anis = []
afs = []
gid_anis = defaultdict(lambda: {})
gid_afs = defaultdict(lambda: {})
all_similar = True
for gid1, gid2 in combinations(type_gids, 2):
ani, af = FastANI.symmetric_ani(ani_af, gid1, gid2)
if ani < 99 or af < 0.65:
all_similar = False
anis.append(ani)
afs.append(af)
gid_anis[gid1][gid2] = ani
gid_anis[gid2][gid1] = ani
gid_afs[gid1][gid2] = af
gid_afs[gid2][gid1] = af
return all_similar, anis, afs, gid_anis, gid_afs
def run(self, cur_gtdb_metadata_file, cur_genomic_path_file,
qc_passed_file, ncbi_genbank_assembly_file, ltp_taxonomy_file,
gtdb_type_strains_ledger, untrustworthy_type_ledger,
ncbi_env_bioproject_ledger):
"""Resolve cases where a species has multiple genomes assembled from the type strain."""
# get species in LTP reference database
self.logger.info(
'Determining species defined in LTP reference database.')
ltp_defined_species = self.ltp_defined_species(ltp_taxonomy_file)
self.logger.info(
f' - identified {len(ltp_defined_species):,} species.')
# create current GTDB genome sets
self.logger.info('Creating current GTDB genome set.')
cur_genomes = Genomes()
cur_genomes.load_from_metadata_file(
cur_gtdb_metadata_file,
gtdb_type_strains_ledger=gtdb_type_strains_ledger,
create_sp_clusters=False,
qc_passed_file=qc_passed_file,
ncbi_genbank_assembly_file=ncbi_genbank_assembly_file,
untrustworthy_type_ledger=untrustworthy_type_ledger,
ncbi_env_bioproject_ledger=ncbi_env_bioproject_ledger)
cur_genomes.load_genomic_file_paths(cur_genomic_path_file)
# parsing genomes manually established to be untrustworthy as type
self.logger.info(
'Determining genomes manually annotated as untrustworthy as type.')
manual_untrustworthy_types = self.parse_untrustworthy_type_ledger(
untrustworthy_type_ledger)
self.logger.info(
f' - identified {len(manual_untrustworthy_types):,} genomes manually annotated as untrustworthy as type.'
)
# Identify NCBI species with multiple genomes assembled from type strain of species. This
# is done using a series of heuristics that aim to ensure that the selected type strain
# genome is reliable. More formal evaluation and a manuscript descirbing this selection
# process is ultimately required. Ideally, the community will eventually adopt a
# database that indicates a single `type genome assembly` for each species instead
# of just indicating a type strain from which many (sometimes dissimilar) assemblies exist.
self.logger.info(
'Determining number of type strain genomes in each NCBI species.')
multi_type_strains_sp = self.sp_with_mult_type_strains(cur_genomes)
self.logger.info(
f' - identified {len(multi_type_strains_sp):,} NCBI species with multiple assemblies indicated as being type strain genomes.'
)
# resolve species with multiple type strain genomes
fout = open(
os.path.join(self.output_dir, 'multi_type_strain_species.tsv'),
'w')
fout.write(
'NCBI species\tNo. type strain genomes\t>=99% ANI\tMean ANI\tStd ANI\tMean AF\tStd AF\tResolution\tGenome IDs\n'
)
fout_genomes = open(
os.path.join(self.output_dir, 'type_strain_genomes.tsv'), 'w')
fout_genomes.write(
'Genome ID\tUntrustworthy\tNCBI species\tGTDB genus\tGTDB species\tLTP species\tConflict with prior GTDB assignment'
)
fout_genomes.write(
'\tMean ANI\tStd ANI\tMean AF\tStd AF\tExclude from RefSeq\tNCBI taxonomy\tGTDB taxonomy\tReason for GTDB untrustworthy as type\n'
)
fout_unresolved = open(
os.path.join(self.output_dir,
'unresolved_type_strain_genomes.tsv'), 'w')
fout_unresolved.write(
'Genome ID\tNCBI species\tGTDB genus\tGTDB species\tLTP species')
fout_unresolved.write(
'\tMean ANI\tStd ANI\tMean AF\tStd AF\tExclude from RefSeq\tNCBI taxonomy\tGTDB taxonomy\n'
)
fout_high_divergence = open(
os.path.join(self.output_dir,
'highly_divergent_type_strain_genomes.tsv'), 'w')
fout_high_divergence.write(
'Genome ID\tNCBI species\tGTDB genus\tGTDB species\tLTP species\tMean ANI\tStd ANI\tMean AF\tStd AF\tExclude from RefSeq\tNCBI taxonomy\tGTDB taxonomy\n'
)
fout_untrustworthy = open(
os.path.join(self.output_dir, 'untrustworthy_type_material.tsv'),
'w')
fout_untrustworthy.write(
'Genome ID\tNCBI species\tGTDB species\tLTP species\tReason for declaring untrustworthy\n'
)
for gid in manual_untrustworthy_types:
ncbi_sp, reason = manual_untrustworthy_types[gid]
fout_untrustworthy.write('{}\t{}\t{}\t{}\t{}\t{}\n'.format(
gid, ncbi_sp, cur_genomes[gid].gtdb_taxa.species,
'<not tested>', 'n/a', 'Manual curation: ' + reason))
processed = 0
num_divergent = 0
unresolved_sp_count = 0
ncbi_ltp_resolved = 0
intra_ani_resolved = 0
ncbi_type_resolved = 0
ncbi_rep_resolved = 0
gtdb_family_resolved = 0
gtdb_genus_resolved = 0
gtdb_sp_resolved = 0
ltp_resolved = 0
# *** Perhaps should be an external flag, but used right now to speed up debugging
use_pickled_results = False
if use_pickled_results:
self.logger.warning(
'Using previously calculated ANI results in: {}'.format(
self.ani_pickle_dir))
prev_gtdb_sp_conflicts = 0
self.logger.info(
'Resolving species with multiple type strain genomes:')
for ncbi_sp, type_gids in sorted(multi_type_strains_sp.items(),
key=lambda kv: len(kv[1])):
assert len(type_gids) > 1
status_str = '-> Processing {} with {:,} type strain genomes [{:,} of {:,} ({:.2f}%)].'.format(
ncbi_sp, len(type_gids), processed + 1,
len(multi_type_strains_sp), (processed + 1) * 100.0 /
len(multi_type_strains_sp)).ljust(128)
sys.stdout.write('{}\r'.format(status_str))
sys.stdout.flush()
processed += 1
# calculate ANI between type strain genomes
all_similar, anis, afs, gid_anis, gid_afs = self.calculate_type_strain_ani(
ncbi_sp, type_gids, cur_genomes, use_pickled_results)
# read LTP metadata for genomes
ltp_metadata = self.parse_ltp_metadata(type_gids, cur_genomes)
untrustworthy_gids = {}
gtdb_resolved_sp_conflict = False
unresolved_species = False
note = 'All type strain genomes have ANI >99% and AF >65%.'
if not all_similar:
note = ''
# need to establish which genomes are untrustworthy as type
num_divergent += 1
unresolved_species = True
# write out highly divergent cases for manual inspection;
# these should be compared to the automated selection
if np_mean(anis) < 95:
for gid in type_gids:
ltp_species = self.ltp_species(gid, ltp_metadata)
fout_high_divergence.write(
'{}\t{}\t{}\t{}\t{}\t{:.2f}\t{:.3f}\t{:.3f}\t{:.4f}\t{}\t{}\t{}\n'
.format(gid, ncbi_sp,
cur_genomes[gid].gtdb_taxa.genus,
cur_genomes[gid].gtdb_taxa.species,
' / '.join(ltp_species),
np_mean(list(gid_anis[gid].values())),
np_std(list(gid_anis[gid].values())),
np_mean(list(gid_afs[gid].values())),
np_std(list(gid_afs[gid].values())),
cur_genomes[gid].excluded_from_refseq_note,
cur_genomes[gid].ncbi_taxa,
cur_genomes[gid].gtdb_taxa))
# filter genomes marked as `untrustworthy as type` at NCBI and where the LTP
# assignment also suggest the asserted type material is incorrect
resolved, untrustworthy_gids = self.resolve_validated_untrustworthy_ncbi_genomes(
gid_anis, ncbi_sp, type_gids, ltp_metadata,
ltp_defined_species, cur_genomes)
if resolved:
note = "Species resolved by removing genomes considered `untrustworthy as type` and with a LTP BLAST hit confirming the assembly is likely untrustworthy"
ncbi_ltp_resolved += 1
# try to resolve by LTP 16S BLAST results
if not resolved:
resolved, untrustworthy_gids = self.resolve_ltp_conflict(
gid_anis, ncbi_sp, type_gids, ltp_metadata, 0)
if resolved:
note = 'Species resolved by identifying conflicting or lack of LTP BLAST results'
ltp_resolved += 1
# try to resolve species using intra-specific ANI test
if not resolved:
resolved, untrustworthy_gids = self.resolve_by_intra_specific_ani(
gid_anis)
if resolved:
note = 'Species resolved by intra-specific ANI test'
intra_ani_resolved += 1
# try to resolve by GTDB family assignment
if not resolved:
resolved, untrustworthy_gids = self.resolve_gtdb_family(
gid_anis, ncbi_sp, type_gids, cur_genomes)
if resolved:
note = 'Species resolved by consulting GTDB family classifications'
gtdb_family_resolved += 1
# try to resolve by GTDB genus assignment
if not resolved:
resolved, untrustworthy_gids = self.resolve_gtdb_genus(
gid_anis, ncbi_sp, type_gids, cur_genomes)
if resolved:
note = 'Species resolved by consulting GTDB genus classifications'
gtdb_genus_resolved += 1
# try to resolve by GTDB species assignment
if not resolved:
resolved, untrustworthy_gids = self.resolve_gtdb_species(
gid_anis, ncbi_sp, type_gids, cur_genomes)
if resolved:
note = 'Species resolved by consulting GTDB species classifications'
gtdb_sp_resolved += 1
# try to resolve by considering genomes annotated as type material at NCBI,
# which includes considering if genomes are marked as untrustworthy as type
if not resolved:
resolved, untrustworthy_gids = self.resolve_by_ncbi_types(
gid_anis, type_gids, cur_genomes)
if resolved:
note = 'Species resolved by consulting NCBI assembled from type metadata'
ncbi_type_resolved += 1
# try to resovle by considering genomes annotated as representative genomes at NCBI
if not resolved:
resolved, untrustworthy_gids = self.resolve_by_ncbi_reps(
gid_anis, type_gids, cur_genomes)
if resolved:
note = 'Species resolved by considering NCBI representative genomes'
ncbi_rep_resolved += 1
if resolved:
unresolved_species = False
# check if type strain genomes marked as trusted or untrusted conflict
# with current GTDB species assignment
untrustworthy_gtdb_sp_match = False
trusted_gtdb_sp_match = False
for gid in type_gids:
gtdb_canonical_epithet = canonical_taxon(
specific_epithet(
cur_genomes[gid].gtdb_taxa.species))
if gtdb_canonical_epithet == specific_epithet(ncbi_sp):
if gid in untrustworthy_gids:
untrustworthy_gtdb_sp_match = True
else:
trusted_gtdb_sp_match = True
if untrustworthy_gtdb_sp_match and not trusted_gtdb_sp_match:
prev_gtdb_sp_conflicts += 1
gtdb_resolved_sp_conflict = True
else:
note = 'Species is unresolved; manual curation is required!'
unresolved_sp_count += 1
if unresolved_species:
for gid in type_gids:
ltp_species = self.ltp_species(gid, ltp_metadata)
fout_unresolved.write(
'{}\t{}\t{}\t{}\t{}\t{:.2f}\t{:.3f}\t{:.3f}\t{:.4f}\t{}\t{}\t{}\n'
.format(gid, ncbi_sp,
cur_genomes[gid].gtdb_taxa.genus,
cur_genomes[gid].gtdb_taxa.species,
' / '.join(ltp_species),
np_mean(list(gid_anis[gid].values())),
np_std(list(gid_anis[gid].values())),
np_mean(list(gid_afs[gid].values())),
np_std(list(gid_afs[gid].values())),
cur_genomes[gid].excluded_from_refseq_note,
cur_genomes[gid].ncbi_taxa,
cur_genomes[gid].gtdb_taxa))
# remove genomes marked as untrustworthy as type at NCBI if one or more potential type strain genomes remaining
ncbi_untrustworthy_gids = set([
gid for gid in type_gids if 'untrustworthy as type' in
cur_genomes[gid].excluded_from_refseq_note
])
if len(type_gids - set(untrustworthy_gids) -
ncbi_untrustworthy_gids) >= 1:
for gid in ncbi_untrustworthy_gids:
untrustworthy_gids[
gid] = "Genome annotated as `untrustworthy as type` at NCBI and there are other potential type strain genomes available"
# report cases where genomes marked as untrustworthy as type at NCBI are being retained as potential type strain genomes
num_ncbi_untrustworthy = len(ncbi_untrustworthy_gids)
for gid in type_gids:
if (gid not in untrustworthy_gids and 'untrustworthy as type'
in cur_genomes[gid].excluded_from_refseq_note):
self.logger.warning(
"Retaining genome {} from {} despite being marked as `untrustworthy as type` at NCBI [{:,} of {:,} considered untrustworthy]."
.format(gid, ncbi_sp, num_ncbi_untrustworthy,
len(type_gids)))
# write out genomes identified as being untrustworthy
for gid, reason in untrustworthy_gids.items():
ltp_species = self.ltp_species(gid, ltp_metadata)
if 'untrustworthy as type' in cur_genomes[
gid].excluded_from_refseq_note:
reason += "; considered `untrustworthy as type` at NCBI"
fout_untrustworthy.write('{}\t{}\t{}\t{}\t{}\n'.format(
gid, ncbi_sp, cur_genomes[gid].gtdb_taxa.species,
' / '.join(ltp_species), reason))
# Sanity check that if the untrustworthy genome has an LTP to only the
# expected species, that all other genomes also have a hit to the
# expected species (or potentially no hit). Otherwise, more consideration
# should be given to the genome with the conflicting LTP hit.
if len(ltp_species) == 1 and ncbi_sp in ltp_species:
other_sp = set()
for test_gid in type_gids:
ltp_species = self.ltp_species(test_gid, ltp_metadata)
if ltp_species and ncbi_sp not in ltp_species:
other_sp.update(ltp_species)
if other_sp:
self.logger.warning(
f'Genome {gid} marked as untrustworthy, but this conflicts with high confidence LTP 16S rRNA assignment.'
)
# write out information about all type genomes
for gid in type_gids:
ltp_species = self.ltp_species(gid, ltp_metadata)
fout_genomes.write(
'{}\t{}\t{}\t{}\t{}\t{}\t{}\t{:.2f}\t{:.3f}\t{:.3f}\t{:.4f}\t{}\t{}\t{}\t{}\n'
.format(gid, gid in untrustworthy_gids, ncbi_sp,
cur_genomes[gid].gtdb_taxa.genus,
cur_genomes[gid].gtdb_taxa.species,
' / '.join(ltp_species), gtdb_resolved_sp_conflict,
np_mean(list(gid_anis[gid].values())),
np_std(list(gid_anis[gid].values())),
np_mean(list(gid_afs[gid].values())),
np_std(list(gid_afs[gid].values())),
cur_genomes[gid].excluded_from_refseq_note,
cur_genomes[gid].ncbi_taxa,
cur_genomes[gid].gtdb_taxa,
untrustworthy_gids.get(gid, '')))
fout.write(
'{}\t{}\t{}\t{:.2f}\t{:.3f}\t{:.3f}\t{:.4f}\t{}\t{}\n'.format(
ncbi_sp, len(type_gids), all_similar, np_mean(anis),
np_std(anis), np_mean(afs), np_std(afs), note,
', '.join(type_gids)))
sys.stdout.write('\n')
fout.close()
fout_unresolved.close()
fout_high_divergence.close()
fout_genomes.close()
fout_untrustworthy.close()
self.logger.info(
f'Identified {num_divergent:,} species with 1 or more divergent type strain genomes.'
)
self.logger.info(
f' - resolved {ncbi_ltp_resolved:,} species by removing NCBI `untrustworthy as type` genomes with a conflicting LTP 16S rRNA classifications.'
)
self.logger.info(
f' - resolved {ltp_resolved:,} species by considering conflicting LTP 16S rRNA classifications.'
)
self.logger.info(
f' - resolved {intra_ani_resolved:,} species by considering intra-specific ANI values.'
)
self.logger.info(
f' - resolved {gtdb_family_resolved:,} species by considering conflicting GTDB family classifications.'
)
self.logger.info(
f' - resolved {gtdb_genus_resolved:,} species by considering conflicting GTDB genus classifications.'
)
self.logger.info(
f' - resolved {gtdb_sp_resolved:,} species by considering conflicting GTDB species classifications.'
)
self.logger.info(
f' - resolved {ncbi_type_resolved:,} species by considering type material designations at NCBI.'
)
self.logger.info(
f' - resolved {ncbi_rep_resolved:,} species by considering RefSeq reference and representative designations at NCBI.'
)
if unresolved_sp_count > 0:
self.logger.warning(
f'There are {unresolved_sp_count:,} unresolved species with multiple type strain genomes.'
)
self.logger.warning(
'These should be handled before proceeding with the next step of GTDB species updating.'
)
self.logger.warning(
"This can be done by manual curation and adding genomes to 'untrustworthy_type_ledger'."
)
self.logger.info(
f'Identified {prev_gtdb_sp_conflicts:,} cases where resolved type strain conflicts with prior GTDB assignment.'
)
class RepActions(object):
"""Perform initial actions required for changed representatives."""
def __init__(self, ani_cache_file, cpus, output_dir):
"""Initialization."""
self.output_dir = output_dir
self.logger = logging.getLogger('timestamp')
self.fastani = FastANI(ani_cache_file, cpus)
# action parameters
self.genomic_update_ani = 99.0
self.genomic_update_af = 0.80
self.new_rep_ani = 99.0
self.new_rep_af = 0.80
self.new_rep_qs_threshold = 10 # increase in ANI score require to select
# new representative
self.action_log = open(os.path.join(self.output_dir, 'action_log.tsv'),
'w')
self.action_log.write(
'Genome ID\tPrevious GTDB species\tAction\tParameters\n')
self.new_reps = {}
def rep_change_gids(self, rep_change_summary_file, field, value):
"""Get genomes with a specific change."""
gids = {}
with open(rep_change_summary_file) as f:
header = f.readline().strip().split('\t')
field_index = header.index(field)
prev_sp_index = header.index('Previous GTDB species')
for line in f:
line_split = line.strip().split('\t')
v = line_split[field_index]
if v == value:
prev_sp = line_split[prev_sp_index]
gids[line_split[0]] = prev_sp
return gids
def top_ani_score_prev_rep(self, prev_rid, sp_cids, prev_genomes,
cur_genomes):
"""Identify genome in cluster with highest balanced ANI score to genomic file of representative in previous GTDB release."""
max_score = -1e6
max_rid = None
max_ani = None
max_af = None
for cid in sp_cids:
ani, af = self.fastani.symmetric_ani_cached(
f'{prev_rid}-P', f'{cid}-C',
prev_genomes[prev_rid].genomic_file,
cur_genomes[cid].genomic_file)
cur_score = cur_genomes[cid].score_ani(ani)
if (cur_score > max_score
or (cur_score == max_score and ani > max_ani)):
max_score = cur_score
max_rid = cid
max_ani = ani
max_af = af
return max_rid, max_score, max_ani, max_af
def top_ani_score(self, prev_rid, sp_cids, cur_genomes):
"""Identify genome in cluster with highest balanced ANI score to representative genome."""
# calculate ANI between representative and genomes in species cluster
gid_pairs = []
for cid in sp_cids:
gid_pairs.append((cid, prev_rid))
gid_pairs.append((prev_rid, cid))
ani_af = self.fastani.pairs(gid_pairs,
cur_genomes.genomic_files,
report_progress=False,
check_cache=True)
# find genome with top ANI score
max_score = -1e6
max_rid = None
max_ani = None
max_af = None
for cid in sp_cids:
ani, af = symmetric_ani(ani_af, prev_rid, cid)
cur_score = cur_genomes[cid].score_ani(ani)
if cur_score > max_score:
max_score = cur_score
max_rid = cid
max_ani = ani
max_af = af
return max_rid, max_score, max_ani, max_af
def get_updated_rid(self, prev_rid):
"""Get updated representative."""
if prev_rid in self.new_reps:
gid, action = self.new_reps[prev_rid]
return gid
return prev_rid
def update_rep(self, prev_rid, new_rid, action):
"""Update representative genome for GTDB species cluster."""
if prev_rid in self.new_reps and self.new_reps[prev_rid][0] != new_rid:
self.logger.warning(
'Representative {} was reassigned multiple times: {} {}.'.
format(prev_rid, self.new_reps[prev_rid], (new_rid, action)))
self.logger.warning(
'Assuming last reassignment of {}: {} has priority.'.format(
new_rid, action))
self.new_reps[prev_rid] = (new_rid, action)
def genomes_in_current_sp_cluster(self, prev_rid, prev_genomes,
new_updated_sp_clusters, cur_genomes):
"""Get genomes in current species cluster."""
assert prev_rid in prev_genomes.sp_clusters
sp_cids = prev_genomes.sp_clusters[prev_rid]
if prev_rid in new_updated_sp_clusters:
sp_cids = sp_cids.union(new_updated_sp_clusters[prev_rid])
sp_cids = sp_cids.intersection(cur_genomes)
return sp_cids
def action_genomic_lost(self, rep_change_summary_file, prev_genomes,
cur_genomes, new_updated_sp_clusters):
"""Handle species with lost representative genome."""
# get genomes with specific changes
self.logger.info(
'Identifying species with lost representative genome.')
genomic_lost_rids = self.rep_change_gids(rep_change_summary_file,
'GENOMIC_CHANGE', 'LOST')
self.logger.info(
f' ... identified {len(genomic_lost_rids):,} genomes.')
# calculate ANI between previous and current genomes
for prev_rid, prev_gtdb_sp in genomic_lost_rids.items():
sp_cids = self.genomes_in_current_sp_cluster(
prev_rid, prev_genomes, new_updated_sp_clusters, cur_genomes)
params = {}
if sp_cids:
action = 'GENOMIC_CHANGE:LOST:REPLACED'
new_rid, top_score, ani, af = self.top_ani_score_prev_rep(
prev_rid, sp_cids, prev_genomes, cur_genomes)
assert (new_rid != prev_rid)
params['new_rid'] = new_rid
params['ani'] = ani
params['af'] = af
params['new_assembly_quality'] = cur_genomes[
new_rid].score_assembly()
params['prev_assembly_quality'] = prev_genomes[
prev_rid].score_assembly()
self.update_rep(prev_rid, new_rid, action)
else:
action = 'GENOMIC_CHANGE:LOST:SPECIES_RETIRED'
self.update_rep(prev_rid, None, action)
self.action_log.write('{}\t{}\t{}\t{}\n'.format(
prev_rid, prev_gtdb_sp, action, params))
def action_genomic_update(self, rep_change_summary_file, prev_genomes,
cur_genomes, new_updated_sp_clusters):
"""Handle representatives with updated genomes."""
# get genomes with specific changes
self.logger.info(
'Identifying representatives with updated genomic files.')
genomic_update_gids = self.rep_change_gids(rep_change_summary_file,
'GENOMIC_CHANGE', 'UPDATED')
self.logger.info(
f' ... identified {len(genomic_update_gids):,} genomes.')
# calculate ANI between previous and current genomes
assembly_score_change = []
for prev_rid, prev_gtdb_sp in genomic_update_gids.items():
# check that genome hasn't been lost which should
# be handled differently
assert prev_rid in cur_genomes
ani, af = self.fastani.symmetric_ani_cached(
f'{prev_rid}-P', f'{prev_rid}-C',
prev_genomes[prev_rid].genomic_file,
cur_genomes[prev_rid].genomic_file)
params = {}
params['ani'] = ani
params['af'] = af
params['prev_ncbi_accession'] = prev_genomes[prev_rid].ncbi_accn
params['cur_ncbi_accession'] = cur_genomes[prev_rid].ncbi_accn
assert prev_genomes[prev_rid].ncbi_accn != cur_genomes[
prev_rid].ncbi_accn
if ani >= self.genomic_update_ani and af >= self.genomic_update_af:
params['prev_assembly_quality'] = prev_genomes[
prev_rid].score_assembly()
params['new_assembly_quality'] = cur_genomes[
prev_rid].score_assembly()
action = 'GENOMIC_CHANGE:UPDATED:MINOR_CHANGE'
d = cur_genomes[prev_rid].score_assembly(
) - prev_genomes[prev_rid].score_assembly()
assembly_score_change.append(d)
else:
sp_cids = self.genomes_in_current_sp_cluster(
prev_rid, prev_genomes, new_updated_sp_clusters,
cur_genomes)
if sp_cids:
new_rid, top_score, ani, af = self.top_ani_score_prev_rep(
prev_rid, sp_cids, prev_genomes, cur_genomes)
if new_rid == prev_rid:
params['prev_assembly_quality'] = prev_genomes[
prev_rid].score_assembly()
params['new_assembly_quality'] = cur_genomes[
prev_rid].score_assembly()
action = 'GENOMIC_CHANGE:UPDATED:RETAINED'
else:
action = 'GENOMIC_CHANGE:UPDATED:REPLACED'
params['new_rid'] = new_rid
params['ani'] = ani
params['af'] = af
params['new_assembly_quality'] = cur_genomes[
new_rid].score_assembly()
params['prev_assembly_quality'] = prev_genomes[
prev_rid].score_assembly()
self.update_rep(prev_rid, new_rid, action)
else:
action = 'GENOMIC_CHANGE:UPDATED:SPECIES_RETIRED'
self.update_rep(prev_rid, None, action)
self.action_log.write('{}\t{}\t{}\t{}\n'.format(
prev_rid, prev_gtdb_sp, action, params))
self.logger.info(
' ... change in assembly score for updated genomes: {:.2f} +/- {:.2f}'
.format(np_mean(assembly_score_change),
np_std(assembly_score_change)))
def action_type_strain_lost(self, rep_change_summary_file, prev_genomes,
cur_genomes, new_updated_sp_clusters):
"""Handle representatives which have lost type strain genome status."""
# get genomes with new NCBI species assignments
self.logger.info(
'Identifying representative that lost type strain genome status.')
ncbi_type_species_lost = self.rep_change_gids(rep_change_summary_file,
'TYPE_STRAIN_CHANGE',
'LOST')
self.logger.info(
f' ... identified {len(ncbi_type_species_lost):,} genomes.')
for prev_rid, prev_gtdb_sp in ncbi_type_species_lost.items():
# check that genome hasn't been lost which should
# be handled differently
assert prev_rid in cur_genomes
sp_cids = self.genomes_in_current_sp_cluster(
prev_rid, prev_genomes, new_updated_sp_clusters, cur_genomes)
prev_rep_score = cur_genomes[prev_rid].score_ani(100)
new_rid, top_score, ani, af = self.top_ani_score(
prev_rid, sp_cids, cur_genomes)
params = {}
params['prev_rid_prev_strain_ids'] = prev_genomes[
prev_rid].ncbi_strain_identifiers
params['prev_rid_cur_strain_ids'] = cur_genomes[
prev_rid].ncbi_strain_identifiers
params['prev_rid_prev_gtdb_type_designation'] = prev_genomes[
prev_rid].gtdb_type_designation
params['prev_rid_cur_gtdb_type_designation'] = cur_genomes[
prev_rid].gtdb_type_designation
params[
'prev_rid_prev_gtdb_type_designation_sources'] = prev_genomes[
prev_rid].gtdb_type_designation_sources
params['prev_rid_cur_gtdb_type_designation_sources'] = cur_genomes[
prev_rid].gtdb_type_designation_sources
if top_score > prev_rep_score:
action = 'TYPE_STRAIN_CHANGE:LOST:REPLACED'
assert (prev_rid != new_rid)
params['new_rid'] = new_rid
params['ani'] = ani
params['af'] = af
params['new_assembly_quality'] = cur_genomes[
new_rid].score_assembly()
params['prev_assembly_quality'] = prev_genomes[
prev_rid].score_assembly()
params['new_rid_strain_ids'] = prev_genomes[
new_rid].ncbi_strain_identifiers
params['new_rid_gtdb_type_designation'] = prev_genomes[
new_rid].gtdb_type_designation
params['new_rid_gtdb_type_designation_sources'] = prev_genomes[
new_rid].gtdb_type_designation_sources
self.update_rep(prev_rid, new_rid, action)
else:
action = 'TYPE_STRAIN_CHANGE:LOST:RETAINED'
self.action_log.write('{}\t{}\t{}\t{}\n'.format(
prev_rid, prev_gtdb_sp, action, params))
def action_domain_change(self, rep_change_summary_file, prev_genomes,
cur_genomes):
"""Handle representatives which have new domain assignments."""
# get genomes with new NCBI species assignments
self.logger.info(
'Identifying representative with new domain assignments.')
domain_changed = self.rep_change_gids(rep_change_summary_file,
'DOMAIN_CHECK', 'REASSIGNED')
self.logger.info(f' ... identified {len(domain_changed):,} genomes.')
for prev_rid, prev_gtdb_sp in domain_changed.items():
action = 'DOMAIN_CHECK:REASSIGNED'
params = {}
params['prev_gtdb_domain'] = prev_genomes[
prev_rid].gtdb_taxa.domain
params['cur_gtdb_domain'] = cur_genomes[prev_rid].gtdb_taxa.domain
self.update_rep(prev_rid, None, action)
self.action_log.write('{}\t{}\t{}\t{}\n'.format(
prev_rid, prev_gtdb_sp, action, params))
def action_improved_rep(self, prev_genomes, cur_genomes,
new_updated_sp_clusters):
"""Check if representative should be replace with higher quality genome."""
self.logger.info(
'Identifying improved representatives for GTDB species clusters.')
num_gtdb_ncbi_type_sp = 0
num_gtdb_type_sp = 0
num_ncbi_type_sp = 0
num_complete = 0
num_isolate = 0
anis = []
afs = []
improved_reps = {}
for idx, (prev_rid,
cids) in enumerate(new_updated_sp_clusters.clusters()):
if prev_rid not in cur_genomes:
# indicates genome has been lost
continue
prev_gtdb_sp = new_updated_sp_clusters.get_species(prev_rid)
statusStr = '-> Processing {:,} of {:,} ({:.2f}%) species [{}: {:,} new/updated genomes].'.format(
idx + 1, len(new_updated_sp_clusters),
float(idx + 1) * 100 / len(new_updated_sp_clusters),
prev_gtdb_sp, len(cids)).ljust(86)
sys.stdout.write('%s\r' % statusStr)
sys.stdout.flush()
# get latest representative of GTDB species clusters as it may
# have been updated by a previous update rule
prev_updated_rid = self.get_updated_rid(prev_rid)
prev_rep_score = cur_genomes[prev_updated_rid].score_ani(100)
new_rid, top_score, ani, af = self.top_ani_score(
prev_updated_rid, cids, cur_genomes)
params = {}
action = None
if top_score > prev_rep_score + self.new_rep_qs_threshold:
assert (prev_updated_rid != new_rid)
if (cur_genomes[prev_updated_rid].is_gtdb_type_strain(
) and cur_genomes[prev_updated_rid].ncbi_taxa.specific_epithet
!= cur_genomes[new_rid].ncbi_taxa.specific_epithet
and self.sp_priority_mngr.has_priority(
cur_genomes, prev_updated_rid, new_rid)):
# GTDB species cluster should not be moved to a different type strain genome
# that has lower naming priority
self.logger.warning(
'Reassignments to type strain genome with lower naming priority is not allowed: {}/{}/{}, {}/{}/{}'
.format(
prev_updated_rid,
cur_genomes[prev_updated_rid].ncbi_taxa.species,
cur_genomes[prev_updated_rid].year_of_priority(),
new_rid, cur_genomes[new_rid].ncbi_taxa.species,
cur_genomes[new_rid].year_of_priority()))
continue
action = 'IMPROVED_REP:REPLACED:HIGHER_QS'
params['new_rid'] = new_rid
params['ani'] = ani
params['af'] = af
params['new_assembly_quality'] = cur_genomes[
new_rid].score_assembly()
params['prev_assembly_quality'] = cur_genomes[
prev_updated_rid].score_assembly()
params['new_gtdb_type_strain'] = cur_genomes[
new_rid].is_gtdb_type_strain()
params['prev_gtdb_type_strain'] = cur_genomes[
prev_updated_rid].is_gtdb_type_strain()
params['new_ncbi_type_strain'] = cur_genomes[
new_rid].is_ncbi_type_strain()
params['prev_ncbi_type_strain'] = cur_genomes[
prev_updated_rid].is_ncbi_type_strain()
anis.append(ani)
afs.append(af)
improvement_list = []
gtdb_type_improv = cur_genomes[new_rid].is_gtdb_type_strain(
) and not cur_genomes[prev_updated_rid].is_gtdb_type_strain()
ncbi_type_improv = cur_genomes[new_rid].is_ncbi_type_strain(
) and not cur_genomes[prev_updated_rid].is_ncbi_type_strain()
if gtdb_type_improv and ncbi_type_improv:
num_gtdb_ncbi_type_sp += 1
improvement_list.append(
'replaced with genome from type strain according to GTDB and NCBI'
)
elif gtdb_type_improv:
num_gtdb_type_sp += 1
improvement_list.append(
'replaced with genome from type strain according to GTDB'
)
elif ncbi_type_improv:
num_ncbi_type_sp += 1
improvement_list.append(
'replaced with genome from type strain according to NCBI'
)
if cur_genomes[new_rid].is_isolate(
) and not cur_genomes[prev_updated_rid].is_isolate():
num_isolate += 1
improvement_list.append('MAG/SAG replaced with isolate')
if cur_genomes[new_rid].is_complete_genome(
) and not cur_genomes[prev_updated_rid].is_complete_genome():
num_complete += 1
improvement_list.append('replaced with complete genome')
if len(improvement_list) == 0:
improvement_list.append(
'replaced with higher quality genome')
params['improvements'] = '; '.join(improvement_list)
self.action_log.write('{}\t{}\t{}\t{}\n'.format(
prev_rid, prev_gtdb_sp, action, params))
improved_reps[prev_rid] = (new_rid, action)
sys.stdout.write('\n')
self.logger.info(
f' ... identified {len(improved_reps):,} species with improved representatives.'
)
self.logger.info(
f' ... {num_gtdb_ncbi_type_sp:,} replaced with GTDB/NCBI genome from type strain.'
)
self.logger.info(
f' ... {num_gtdb_type_sp:,} replaced with GTDB genome from type strain.'
)
self.logger.info(
f' ... {num_ncbi_type_sp:,} replaced with NCBI genome from type strain.'
)
self.logger.info(
f' ... {num_isolate:,} replaced MAG/SAG with isolate.')
self.logger.info(
f' ... {num_complete:,} replaced with complete genome assembly.')
self.logger.info(
f' ... ANI = {np_mean(anis):.2f} +/- {np_std(anis):.2f}%; AF = {np_mean(afs)*100:.2f} +/- {np_std(afs)*100:.2f}%.'
)
return improved_reps
def action_naming_priority(self, prev_genomes, cur_genomes,
new_updated_sp_clusters):
"""Check if representative should be replace with genome with higher nomenclatural priority."""
self.logger.info(
'Identifying genomes with naming priority in GTDB species clusters.'
)
out_file = os.path.join(self.output_dir, 'update_priority.tsv')
fout = open(out_file, 'w')
fout.write(
'NCBI species\tGTDB species\tRepresentative\tStrain IDs\tRepresentative type sources\tPriority year\tGTDB type species\tGTDB type strain\tNCBI assembly type'
)
fout.write(
'\tNCBI synonym\tGTDB synonym\tSynonym genome\tSynonym strain IDs\tSynonym type sources\tPriority year\tGTDB type species\tGTDB type strain\tSynonym NCBI assembly type'
)
fout.write('\tANI\tAF\tPriority note\n')
num_higher_priority = 0
assembly_score_change = []
anis = []
afs = []
for idx, prev_rid in enumerate(prev_genomes.sp_clusters):
# get type strain genomes in GTDB species cluster, including genomes new to this release
type_strain_gids = [
gid for gid in prev_genomes.sp_clusters[prev_rid]
if gid in cur_genomes
and cur_genomes[gid].is_effective_type_strain()
]
if prev_rid in new_updated_sp_clusters:
new_type_strain_gids = [
gid for gid in new_updated_sp_clusters[prev_rid]
if cur_genomes[gid].is_effective_type_strain()
]
type_strain_gids.extend(new_type_strain_gids)
if len(type_strain_gids) == 0:
continue
# check if representative has already been updated
updated_rid = self.get_updated_rid(prev_rid)
type_strain_sp = set([
cur_genomes[gid].ncbi_taxa.species for gid in type_strain_gids
])
if len(type_strain_sp) == 1 and updated_rid in type_strain_gids:
continue
updated_sp = cur_genomes[updated_rid].ncbi_taxa.species
highest_priority_gid = updated_rid
if updated_rid not in type_strain_gids:
highest_priority_gid = None
if updated_sp in type_strain_sp:
sp_gids = [
gid for gid in type_strain_gids
if cur_genomes[gid].ncbi_taxa.species == updated_sp
]
hq_gid = select_highest_quality(sp_gids, cur_genomes)
highest_priority_gid = hq_gid
#self.logger.warning('Representative is a non-type strain genome even though type strain genomes exist in species cluster: {}: {}, {}: {}'.format(
# prev_rid, cur_genomes[prev_rid].is_effective_type_strain(), updated_rid, cur_genomes[updated_rid].is_effective_type_strain()))
#self.logger.warning('Type strain genomes: {}'.format(','.join(type_strain_gids)))
# find highest priority genome
for sp in type_strain_sp:
if sp == updated_sp:
continue
# get highest quality genome from species
sp_gids = [
gid for gid in type_strain_gids
if cur_genomes[gid].ncbi_taxa.species == sp
]
hq_gid = select_highest_quality(sp_gids, cur_genomes)
if highest_priority_gid is None:
highest_priority_gid = hq_gid
else:
highest_priority_gid, note = self.sp_priority_mngr.priority(
cur_genomes, highest_priority_gid, hq_gid)
# check if representative should be updated
if highest_priority_gid != updated_rid:
num_higher_priority += 1
ani, af = self.fastani.symmetric_ani_cached(
updated_rid, highest_priority_gid,
cur_genomes[updated_rid].genomic_file,
cur_genomes[highest_priority_gid].genomic_file)
anis.append(ani)
afs.append(af)
d = cur_genomes[highest_priority_gid].score_assembly(
) - cur_genomes[updated_rid].score_assembly()
assembly_score_change.append(d)
action = 'NOMENCLATURE_PRIORITY:REPLACED'
params = {}
params['prev_ncbi_species'] = cur_genomes[
updated_rid].ncbi_taxa.species
params['prev_year_of_priority'] = cur_genomes[
updated_rid].year_of_priority()
params['new_ncbi_species'] = cur_genomes[
highest_priority_gid].ncbi_taxa.species
params['new_year_of_priority'] = cur_genomes[
highest_priority_gid].year_of_priority()
params['new_rid'] = highest_priority_gid
params['ani'] = ani
params['af'] = af
params['priority_note'] = note
self.update_rep(prev_rid, highest_priority_gid, action)
self.action_log.write('{}\t{}\t{}\t{}\n'.format(
prev_rid, cur_genomes[updated_rid].gtdb_taxa.species,
action, params))
fout.write('{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}'.format(
cur_genomes[highest_priority_gid].ncbi_taxa.species,
cur_genomes[highest_priority_gid].gtdb_taxa.species,
highest_priority_gid, ','.join(
sorted(
cur_genomes[highest_priority_gid].strain_ids())),
','.join(
sorted(cur_genomes[highest_priority_gid].
gtdb_type_sources())).upper().replace(
'STRAININFO', 'StrainInfo'),
cur_genomes[highest_priority_gid].year_of_priority(),
cur_genomes[highest_priority_gid].is_gtdb_type_species(),
cur_genomes[highest_priority_gid].is_gtdb_type_strain(),
cur_genomes[highest_priority_gid].ncbi_type_material))
fout.write('\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}\t{}'.format(
cur_genomes[updated_rid].ncbi_taxa.species,
cur_genomes[updated_rid].gtdb_taxa.species, updated_rid,
','.join(sorted(cur_genomes[updated_rid].strain_ids())),
','.join(
sorted(cur_genomes[updated_rid].gtdb_type_sources())
).upper().replace('STRAININFO', 'StrainInfo'),
cur_genomes[updated_rid].year_of_priority(),
cur_genomes[updated_rid].is_gtdb_type_species(),
cur_genomes[updated_rid].is_gtdb_type_strain(),
cur_genomes[updated_rid].ncbi_type_material))
fout.write('\t{:.3f}\t{:.4f}\t{}\n'.format(ani, af, note))
fout.close()
self.logger.info(
f' ... identified {num_higher_priority:,} species with representative changed to genome with higher nomenclatural priority.'
)
self.logger.info(
' ... change in assembly score for new representatives: {:.2f} +/- {:.2f}'
.format(np_mean(assembly_score_change),
np_std(assembly_score_change)))
self.logger.info(' ... ANI: {:.2f} +/- {:.2f}'.format(
np_mean(anis), np_std(anis)))
self.logger.info(' ... AF: {:.2f} +/- {:.2f}'.format(
np_mean(afs), np_std(afs)))
def write_updated_clusters(self, prev_genomes, cur_genomes, new_reps,
new_updated_sp_clusters, out_file):
"""Write out updated GTDB species clusters."""
self.logger.info(
'Writing updated GTDB species clusters to file: {}'.format(
out_file))
fout = open(out_file, 'w')
fout.write(
'Representative genome\tGTDB species\tNo. clustered genomes\tClustered genomes\n'
)
cur_genome_set = set(cur_genomes)
num_clusters = 0
for idx, prev_rid in enumerate(prev_genomes.sp_clusters):
new_rid, action = new_reps.get(prev_rid, [prev_rid, None])
if new_rid is None:
continue
sp_cids = self.genomes_in_current_sp_cluster(
prev_rid, prev_genomes, new_updated_sp_clusters,
cur_genome_set)
fout.write('{}\t{}\t{}\t{}\n'.format(
new_rid, prev_genomes.sp_clusters.get_species(prev_rid),
len(sp_cids), ','.join(sp_cids)))
num_clusters += 1
fout.close()
self.logger.info(f' ... wrote {num_clusters:,} clusters.')
def run(self, rep_change_summary_file, prev_gtdb_metadata_file,
prev_genomic_path_file, cur_gtdb_metadata_file,
cur_genomic_path_file, uba_genome_paths, genomes_new_updated_file,
qc_passed_file, gtdbtk_classify_file, ncbi_genbank_assembly_file,
untrustworthy_type_file, gtdb_type_strains_ledger,
sp_priority_ledger):
"""Perform initial actions required for changed representatives."""
# create previous and current GTDB genome sets
self.logger.info('Creating previous GTDB genome set.')
prev_genomes = Genomes()
prev_genomes.load_from_metadata_file(
prev_gtdb_metadata_file,
gtdb_type_strains_ledger=gtdb_type_strains_ledger,
uba_genome_file=uba_genome_paths,
ncbi_genbank_assembly_file=ncbi_genbank_assembly_file,
untrustworthy_type_ledger=untrustworthy_type_file)
self.logger.info(
' ... previous genome set has {:,} species clusters spanning {:,} genomes.'
.format(len(prev_genomes.sp_clusters),
prev_genomes.sp_clusters.total_num_genomes()))
self.logger.info('Creating current GTDB genome set.')
cur_genomes = Genomes()
cur_genomes.load_from_metadata_file(
cur_gtdb_metadata_file,
gtdb_type_strains_ledger=gtdb_type_strains_ledger,
create_sp_clusters=False,
uba_genome_file=uba_genome_paths,
qc_passed_file=qc_passed_file,
ncbi_genbank_assembly_file=ncbi_genbank_assembly_file,
untrustworthy_type_ledger=untrustworthy_type_file)
self.logger.info(
f' ... current genome set contains {len(cur_genomes):,} genomes.')
# get path to previous and current genomic FASTA files
self.logger.info(
'Reading path to previous and current genomic FASTA files.')
prev_genomes.load_genomic_file_paths(prev_genomic_path_file)
prev_genomes.load_genomic_file_paths(uba_genome_paths)
cur_genomes.load_genomic_file_paths(cur_genomic_path_file)
cur_genomes.load_genomic_file_paths(uba_genome_paths)
# created expanded previous GTDB species clusters
new_updated_sp_clusters = SpeciesClusters()
self.logger.info(
'Creating species clusters of new and updated genomes based on GTDB-Tk classifications.'
)
new_updated_sp_clusters.create_expanded_clusters(
prev_genomes.sp_clusters, genomes_new_updated_file, qc_passed_file,
gtdbtk_classify_file)
self.logger.info(
'Identified {:,} expanded species clusters spanning {:,} genomes.'.
format(len(new_updated_sp_clusters),
new_updated_sp_clusters.total_num_genomes()))
# initialize species priority manager
self.sp_priority_mngr = SpeciesPriorityManager(sp_priority_ledger)
# take required action for each changed representatives
self.action_genomic_lost(rep_change_summary_file, prev_genomes,
cur_genomes, new_updated_sp_clusters)
self.action_genomic_update(rep_change_summary_file, prev_genomes,
cur_genomes, new_updated_sp_clusters)
self.action_type_strain_lost(rep_change_summary_file, prev_genomes,
cur_genomes, new_updated_sp_clusters)
self.action_domain_change(rep_change_summary_file, prev_genomes,
cur_genomes)
if True: #***
improved_reps = self.action_improved_rep(prev_genomes, cur_genomes,
new_updated_sp_clusters)
pickle.dump(
improved_reps,
open(os.path.join(self.output_dir, 'improved_reps.pkl'), 'wb'))
else:
self.logger.warning(
'Reading improved_reps for pre-cached file. Generally used only for debugging.'
)
improved_reps = pickle.load(
open(os.path.join(self.output_dir, 'improved_reps.pkl'), 'rb'))
for prev_rid, (new_rid, action) in improved_reps.items():
self.update_rep(prev_rid, new_rid, action)
self.action_naming_priority(prev_genomes, cur_genomes,
new_updated_sp_clusters)
# report basic statistics
num_retired_sp = sum(
[1 for v in self.new_reps.values() if v[0] is None])
num_replaced_rids = sum(
[1 for v in self.new_reps.values() if v[0] is not None])
self.logger.info(f'Identified {num_retired_sp:,} retired species.')
self.logger.info(
f'Identified {num_replaced_rids:,} species with a modified representative genome.'
)
self.action_log.close()
# write out representatives for existing species clusters
fout = open(os.path.join(self.output_dir, 'updated_species_reps.tsv'),
'w')
fout.write(
'Previous representative ID\tNew representative ID\tAction\tRepresentative status\n'
)
for rid in prev_genomes.sp_clusters:
if rid in self.new_reps:
new_rid, action = self.new_reps[rid]
if new_rid is not None:
fout.write(f'{rid}\t{new_rid}\t{action}\tREPLACED\n')
else:
fout.write(f'{rid}\t{new_rid}\t{action}\tLOST\n')
else:
fout.write(f'{rid}\t{rid}\tNONE\tUNCHANGED\n')
fout.close()
# write out updated species clusters
out_file = os.path.join(self.output_dir, 'updated_sp_clusters.tsv')
self.write_updated_clusters(prev_genomes, cur_genomes, self.new_reps,
new_updated_sp_clusters, out_file)