def get_metagenome_cluster_prokka(prokka_dir=None, dest_dir=None, suffix='ffn'):
"""copy all cluster 'ffn' files from remote directory.
Then prepend file name on froent of header lines,
and replace all spaces in lines with underscores '_'
Note: *_dir args should be 'APath' instances
"""
#TODO: ensure all files named after their cluster (w/o _,-..?) !!
srce_dir = prokka_dir or APath(CONFIG.get('paths').get('prokka_dir'))
dest_dir = dest_dir or APath(CONFIG.get('paths').get('working_dir'))
log.info('Copying and processing Prokka ffn files '
'from {srce_dir} into {dest_dir}')
dest_files = []
assert next(srce_dir.glob('*'+suffix)), f'No matching files in the dir "{srce_dir.abspath}"'
for ffn in srce_dir.glob('*'+suffix):
log.info(f'Copying {ffn.name}')
try:
dst_fn = dest_dir / ffn.name
dest_files.append(shutil.copyfile(ffn, dst_fn))
log.info(f'Prepending "{ffn.stem}" into sequence headers')
sed_inplace(dst_fn, r'^>', f'>{ffn.stem}_')
replace_spaces(dst_fn, '_')
except IOError as e:
log.error(f'IOError, copying "{e.filename}" to "{e.filename2}": {e}')
raise e
except Exception as e:
log.error(f'Error: {e}')
raise e
return dest_files
def generate_musicc_regex(musiccs=None, begin_regex=None):
"""Generate regex pattern for matching MUSiCC patterns.
List of patterns can be passed or is read from config file.
'begin_regex' can be character class or other `re` at beginning of pattern.
"""
try:
musiccs = musiccs or CONFIG.get('filters').get('musicc_list')
bgn = begin_regex or CONFIG.get('filters').get('begin_regex')
log.debug(f'MUSiCC check list: "{musiccs}"')
mpatt = f'{bgn}(' +'|'.join(musiccs)+ ')'
log.debug(f'MUSiCC check pattern: "{mpatt}"')
muser = re.compile(mpatt)
return muser
except Exception as e:
log.error(f'Generating MUSiCC regex match: {e}')
raise e
def filter_probe_seqs(dbname, cluster_id, table_name=None):
"""Create db view onto blast results table, limiting on (below default values):
- dupes
- pct_identity
- within GC min>max
- =40bp length
- hit on this clust
- not match tRNA names
"""
try:
log.info(f'Filtering headers in db view for {dbname}')
db = dbname or DB_CFG.get('name')
table_name = table_name or DB_CFG.get('probes_table').get('name')
filter_view = DB_CFG.get('probes_view').get('name')
field_list = DB_CFG.get('probes_view').get('cols').copy()
field_sql = ', '.join(field_list)
gc_min = CONFIG.get('gc_percent').get('min_percent')
gc_max = CONFIG.get('gc_percent').get('max_percent')
probe_length = CONFIG.get('catch').get('probe_length')
pct_identity = CONFIG.get('filters').get('pct_identity')
trna_list = CONFIG.get('filters').get('trna_list')
trna_wheres = [ f'sseqid NOT LIKE "%{t}%"' for t in trna_list ]
trna_where_def = ' AND ('+ ' AND '.join(trna_wheres) +')'
wheres = [f'gc_pct between "{gc_min}" and "{gc_max}"',
f'pident={pct_identity}',
f'length={probe_length}',
f'qseqid like "{cluster_id}%"',
] + trna_wheres
where_def = ' AND '.join(wheres) + trna_where_def
group_def = 'qseqid HAVING count(qseqid)=1'
ddl_view = f'DROP VIEW IF EXISTS {filter_view};'
ddl_view = (f'CREATE VIEW {filter_view} AS'
f' SELECT {field_sql} FROM {table_name}'
f' WHERE {where_def} GROUP BY {group_def};')
# log.debug(f'filtering view query: "{ddl_view}"')
create_success = Sdb.exec_ddl(db, ddl_view)
return create_success
except Exception as e:
log.error(f'Writing to db "{db}": {e}')
raise e
def export_final_sets(dbname, cluster_id, final_probe_amount=1, randomly=True):
"""Export final sets of (possibly random) probe sequences into fasta format;
one file for 'musicc', one for non.
"""
log.info(f'Exporting probes for {cluster_id}')
working_dir = APath(CONFIG.get('paths').get('working_dir'))
final_amount = int(final_probe_amount) or int(CONFIG.get('general').get('final_probe_amount'))
random_picks = randomly or CONFIG.get('general').get('final_probe_random')
filter_view = DB_CFG.get('probes_view').get('name')
"""final_fields taken from config/database/probes_view_cols last words (post-space)"""
final_fields = [col.split(' ')[-1] for col in DB_CFG.get('probes_view').get('cols').copy()]
for which, where in (('normal','0'), ('musicc','1')):
export_bits = '.'.join([cluster_id, 'probes', 'final', which, 'fasta'])
export_file = working_dir / export_bits
whim = 'is_musicc='+where
record_count = Sdb.iter_select(dbname, filter_view, where=whim, fields='count(*) as recs')
record_count = next(record_count).pop('recs')
log.debug(f' ... record_count: {record_count}')
if record_count == 0:
log.notice(f'No filtered "{which}" probes for cluster "{cluster_id}".')
write_out_file('', export_file, mode='a') # write empty file
next
if random_picks:
final_amount = final_amount if record_count >= final_amount else record_count
row_nums = random.sample(range(record_count), k=final_amount)
else:
row_nums = [r for r in range(final_amount)]
log.debug(f' ... row_nums: {row_nums}')
probes_selector = Sdb.iter_select(dbname, filter_view, where=whim, fields=final_fields)
log.info(f'Exporting to file {export_file}')
for row in [row for num, row in enumerate(probes_selector) if num in row_nums]:
seq = row.pop('probe_seq') # NB: presumption of column name 'probe_seq' in filter view!!
head = '>' + '|'.join([str(v) for v in row.values()])
probe_fasta = os.linesep.join([head, seq, '']) # final '' elem appends EOL
log.debug(f' ... writing to file {export_file}: "{probe_fasta}"')
write_out_file(probe_fasta, export_file, mode='a')
def catch_design_probes(gbin, dest_dir=None, reuse_existing=False):
"""Design cluster probes using catch app.
Prepend cluster gbin name into header in resulting sequence files.
Requires: [catch]
Note: file, dir args should be 'APath' instances
"""
log.info(f'Designing probes for {gbin.name}')
dest_dir = dest_dir or APath(CONFIG.get('paths').get('working_dir'))
# log.notice(f'reuse_existing: {reuse_existing}')
try:
catch_app = CONFIG.get('APPS').get('catch')
# insert '.probes' into outfile and log names
probe_out = dest_dir / '.'.join([gbin.stem, 'probes', gbin.suffix[1:]])
catch_tsv = dest_dir / f'{gbin.stem}.probe_coverage_analysis.tsv'
if reuse_existing and probe_out.exists():
log.info(f'Using pre-existing cluster probes file "{probe_out}"')
return probe_out
opt_probe_length = str(CONFIG.get('catch').get('probe_length'))
opt_probe_stride = str(CONFIG.get('catch').get('probe_stride'))
cmd = [catch_app,
'--write-analysis-to-tsv', catch_tsv.abspath,
'--probe-length', opt_probe_length,
'--probe-stride', opt_probe_stride,
'--output-probes', probe_out.abspath,
gbin.abspath,
]
output = run_cmd(cmd)
log.info(f'Prepending clusterID to seq headers in {probe_out}')
sed_inplace(probe_out, r'^>', f'>{gbin.stem}_')
except Exception as e:
log.error(f'Error: {e}')
raise e
else:
return probe_out
def blast_clust_probes_on_genome(probe_file, blastdb):
"""Run 'blastn' of cluster's probe fasta on genome blastdb.
Note: probe_file be 'APath' instance, blastdb param is string of filename or filepath.
"""
log.info(f'Blasting cluster\'s probes ({probe_file}) on genome db {blastdb}')
try:
blastn = CONFIG.get('APPS').get('blastn')
dust = CONFIG.get('blastn').get('dust', 'no')
evalue = CONFIG.get('blastn').get('evalue', '10')
numaln = CONFIG.get('blastn').get('num_alignments', '250')
numcpu = CONFIG.get('blastn').get('num_threads', '1')
outfmt = CONFIG.get('blastn').get('outfmt', '10')
fields = DB_CFG.get('blastn').get('fields').copy()
extras = CONFIG.get('blastn').get('fields')
fields += [f for f in extras if f not in fields]
field_fmt = ' '.join(fields)
if not probe_file.is_file():
err_msg = f'Path: "{probe_file.abspath}" is not a file?!'
log.warning(err_msg)
return err_msg
cmd = [blastn,
'-task', 'blastn',
'-query', probe_file.abspath,
'-db', blastdb,
'-dust', dust,
'-evalue', evalue,
'-num_alignments', numaln,
'-num_threads', numcpu,
'-outfmt', f'{outfmt} {field_fmt}',
]
output = run_cmd(cmd, only_stdout=True)
log.notice('blast output: '+output[0:100])
"""blast_rows is rows of all output: here conv'd to list of list-per-line"""
blast_rows = [ row.split(',') for row in output.splitlines() ]
# log.notice(f'show blast_rows[0]: {blast_rows[0]}')
log.info(f'Number of blast matches: {len(blast_rows)}')
except Exception as e:
log.error(f'Error: {e}')
raise e
else:
return blast_rows
def makeblastdb(fastaname, blast_db=None):
"""make blast db from fasta file
Requires: [makeblastdb]
"""
log.info(f'Making blastdb for {fastaname}')
try:
dest_db = blast_db or fastaname
mkblastdb = CONFIG.get('APPS').get('blastdb')
cmd = [mkblastdb,
'-dbtype', 'nucl',
'-in', fastaname,
'-out', dest_db,
'-logfile', fastaname+'.makeblastdb.log'
]
output = run_cmd(cmd)
except Exception as e:
log.error(f'Error: {e}')
raise e
else:
return output
def import_blasts_to_db(blast_hit_list, db_name=None, table_name=None):
"""Import blast results to database."""
"""check args or use config options"""
db = db_name or DB_CFG.get('clusterdb').get('name')
table_name = table_name or DB_CFG.get('probes_table').get('name')
table_cols = DB_CFG.get('probes_table').get('cols')
index_cols = ', '.join(table_cols.keys()) # index only the default columns
"""add in extra non-default blastn fields to the column list without datatype"""
blastn_fields = CONFIG.get('blastn').get('fields').copy()
for fld in blastn_fields:
if fld not in table_cols:
table_cols[fld] = '' # empty datatypes
col_defs = ', '.join([' '.join(t) for t in table_cols.items()])
ddl_table = f'CREATE TABLE IF NOT EXISTS {table_name} ({col_defs});'
create_table = Sdb.exec_ddl(db, ddl_table)
ddl_index = f'CREATE INDEX IF NOT EXISTS "probes_idx" ON {table_name} ({index_cols});'
create_index = Sdb.exec_ddl(db, ddl_index)
import_success = Sdb.import_data(blast_hit_list, db, table=table_name)
return create_table and create_index and import_success
def main_pipe(*, config_file:'c'=None, debug=False):
"""Execute the steps of the targeted probe design pipeline
:param config_file: non-default TOML configuration file to set modified options.
:param debug: show internal debugging messages and configuration.
"""
try:
log.name = 'Targeted_Pipeline'
log.info('Beginning execution of the targeted design probe pipeline.')
if debug:
log.level_name = 'DEBUG'
for lh in log.handlers:
lh.level_name = 'DEBUG'
if config_file:
log.name = 'Targeted:Read Config Options'
user_cfg = read_config_file(config_file)
for k in CONFIG:
if k in user_cfg:
CONFIG[k].update(user_cfg[k])
else:
log.notice('Using default configuration. (Install module "clize" for command args.)')
log.name = 'Targeted:Check Config Options'
check_options()
log.name = 'Targeted_Pipeline'
working_dir = APath(CONFIG.get('paths').get('working_dir'))
gbin_dir = APath(CONFIG.get('paths').get('genome_bins'))
gbin_suff = CONFIG.get('general').get('genome_bins_suffix')
"""Make blast dbs for all ffn, if no preexisting designated use_blastdb"""
log.name = 'Targeted:blastdb'
use_blastdb = CONFIG.get('paths').get('use_blastdb', None)
if use_blastdb:
try:
use_blastdb_path = APath(use_blastdb)
blastdb_name = use_blastdb_path.name
with use_blastdb_path.resolve(strict=True):
log.info(f'Using pre-existing blastdb: {use_blastdb_path.abspath}')
blast_all_clusters = use_blastdb_path.abspath
prokka_files = [] # for final cleanup
except Exception as e:
log.error(f'Unable to use pre-existing blastdb: {use_blastdb}')
raise e
else:
blastdb_name = DB_CFG.get('blastdb').get('name')
blastdb_path = working_dir / blastdb_name
try:
"""Copy cluster prediction files and make blast dbs for each"""
# log.name = 'Targeted:GetMwgsProkka'
prokka_dir = APath(CONFIG.get('paths').get('prokka_dir'))
prokka_suff = CONFIG.get('general').get('prokka_prediction_suffix')
prokka_files = get_metagenome_cluster_prokka(prokka_dir, working_dir, suffix=prokka_suff)
log.info(f'Creating blastdb: {blastdb_path.abspath}')
"""concat all clusters' prokka_files into one for blasting"""
blast_all_clusters = concatenate_files(
working_dir.abspath,
blastdb_path.abspath,
suffix=prokka_suff,
clobber=True
)
makeblastdb(blast_all_clusters)
except Exception as e:
log.error(f'Unable to create blastdb: {blastdb_name}')
raise e
"""Design probes for genome bin fastas"""
#TODO: run in parallel, use multiprocessing.Pool ??
probe_fastas = []
for gbin in gbin_dir.glob('*'+gbin_suff):
log.name = 'Targeted Pipeline'
probe_file = targeted_genome_bin_probes(gbin, blastdb=blast_all_clusters)
probe_fastas.append(probe_file)
except Exception as e:
log.error(f'Error. {e.args}')
raise e
else:
log.name = 'Targeted Pipeline'
log.notice(f'''Completed this run of targeted probe pipeline!
\nConfig options used: {tomlkit.dumps(CONFIG)}''')
if debug:
log.notice(f'''\nDatabase Config options used: {tomlkit.dumps(DB_CFG)}''')
log.info('Finalize by tidying up intermediate files.')
finalize_outfiles(working_dir,
blastdb=blastdb_name,
annots=prokka_files,
probes=probe_fastas)
def finalize_outfiles(working_dir='', blastdb=None, annots=[], probes=[]):
"""Check CONFIG settings, delete or compress the intermediate files, then compress logs.
:param working_dir: string of path to work in
:param blastdb: name of blastdb created
:param annote: list of modified annotation/prediction files
:param probes: list of intial probe fastas created by 'catch'
"""
log.name = 'Finalizing'
if not working_dir:
working_dir = os.cwd()
keepers = CONFIG.get('general').get('keep_files').copy()
compress = CONFIG.get('general').get('compress_files')
file_globs = config.TMP_FILE_GLOBS.copy()
# special cases of files tracked without
argfiles =dict(
blast_db = blastdb,
annotation_mods = [p.abspath for p in annots],
catch_probes = [p.abspath for p in probes],
)
if CONFIG.get('paths').get('use_blastdb', None):
try:
log.info('Keeping blastdb')
for ftype in ['blast_db', 'annotation_mods']:
file_globs.pop(ftype)
argfiles.pop(ftype)
except ValueError as e:
log.error(e)
for ftype, flist in argfiles.items():
try:
if ftype in keepers:
log.info(f'Tidying up {ftype}')
file_globs.pop(ftype)
tidy_up_files(flist, working_dir, True, compress)
except Exception:
pass
if 'target_dbs' in keepers:
db_name = file_globs.get('target_dbs')
dbs_glob = '_'.join(['*', db_name])
try:
log.info('Vacuuming databases')
for db in APath(working_dir).glob(dbs_glob):
Sdb.exec_ddl(db.abspath, 'VACUUM;')
log.debug(f'Vacuumed database: {db}')
except Exception:
pass
for k, glb in file_globs.items():
log.info(f'Tidying up {k}')
glob = ''.join(['*', glb]) if glb else argfiles[k]
if k in keepers:
tidy_up_files(glob, working_dir, True, compress)
else:
tidy_up_files(glob, working_dir, keep=False)
# ...and finally compress the logfile
gzip_compress(log.filename)
def check_options():
"""check validity of CONFIG settings, try setup if needed"""
# Check [paths] options, either it exists or create it:
try:
log.info('Checking files and directories.')
paths = [ 'working_dir', 'genome_bins', 'use_blastdb', 'prokka_dir' ]
path_opts = CONFIG.get('paths')
path = 'working_dir'
log.info(f'Checking "{path}"')
ppath = APath(path_opts.get(path))
if ppath.is_dir():
log.info(f'Path: "{ppath.name}" directory found.')
else:
log.warning(f'Path for "{ppath.name}" directory not found!')
try:
ppath.mkdir(parents=True, exist_ok=True)
log.notice(f'Path: "{ppath.abspath}" directory created.')
except FileExistsError as e:
log.error('File/dir exists.')
raise e
path_opts[path] = ppath.abspath
path = 'genome_bins'
log.info(f'Checking "{path}"')
ppath = APath(path_opts.get(path), '')
assert ppath.is_dir(), f'Path "{ppath}" is not found!'
log.info(f'Path: "{ppath.abspath}" file found.')
path_opts[path] = ppath.abspath
path = 'use_blastdb'
ppath = path_opts.get(path)
if ppath:
log.info(f'Checking "{path}"')
ppath = APath(path_opts.get(path), '')
assert ppath.is_file(), f'Path "{ppath}" is not a file!'
log.info(f'Path: "{ppath.abspath}" file found.')
path_opts[path] = ppath.abspath
else:
path = 'prokka_dir'
log.info(f'Checking "{path}"')
ppath = APath(path_opts.get(path), '')
assert ppath.is_dir(), f'Path "{ppath}" is not found!'
log.info(f'Path: "{ppath.abspath}" directory found.')
path_opts[path] = ppath.abspath
except AssertionError as e:
log.error(e)
sys.exit(1)
raise e
except Exception as e:
log.error(e)
sys.exit(1)
raise e
# APP executable checks:
apps = CONFIG.get('APPS')
cmd_exists = lambda x: shutil.which(x) is not None
try:
log.info('Checking applications usable.')
log.debug(f'PATH=\"{os.environ.get("PATH")}\"')
for opt, app in apps.items():
log.notice(f'App for: "{opt}"')
if cmd_exists(app):
log.info(f'App: "{app}" found.')
else:
log.warning(f'App: "{app}" is not found?!')
except Exception as e:
raise e
def targeted_genome_bin_probes(genome_bin, blastdb=None):
"""Generate, process, filter and export probes for a cluster genome bin"""
log.notice(f'Generating targeted probes for genome bin: {genome_bin.name}')
working_dir = APath(CONFIG.get('paths').get('working_dir'))
blast_header = DB_CFG.get('blastn').get('fields').copy()
blast_extras = CONFIG.get('blastn').get('fields').copy()
"""add in extra non-default blastn fields to the header"""
for fld in blast_extras:
if fld not in blast_header:
blast_header.append(fld)
blast_header.extend([ 'gc_pct', 'is_musicc' ])
blastdb = blastdb or makeblastdb(genome_bin)
cluster_id = genome_bin.stem
log.name = 'Probe:CatchDesign'
reuse_existing_probes = CONFIG.get('catch').get('reuse_existing_probe_files')
probes_file = catch_design_probes(genome_bin, reuse_existing=reuse_existing_probes)
"""probe_blasts is list of all blast matched records (as lists)"""
log.name = 'Probes:Blast'
probe_blasts = blast_clust_probes_on_genome(probes_file, blastdb)
"""Calculate GC% for each seq in probes. Append that and seq onto probe_blasts"""
probe_ids = set( [pb[0] for pb in probe_blasts] )
probes_gc = {}
log.name = ('Probe:GC,MUSiCC')
log.info('Processing blast match sequences for GC%, and the seq hits for MUSiCC')
musicc_re = generate_musicc_regex()
for header, seq in read_fasta(probes_file):
qid = header.replace('>','')
if qid in probe_ids:
log.info(f'Processing probe seq id: "{qid}"')
for pb in probe_blasts:
if pb[0] == qid:
if pb[0] not in probes_gc:
log.debug(f' ... Calc GC% on "{qid}"')
probes_gc[pb[0]] = pct_gc(seq)
pb.append( probes_gc[pb[0]] )
# log.debug(f' ... Check MUSiCC on "{pb[1]}"')
is_musicc = 1 if musicc_re.search(pb[1]) else 0
pb.append( is_musicc )
"""Get list of fields; write to csv file as header"""
probe_blasts.insert(0, blast_header)
blast_probe_file = probes_file.with_suffix('.blasts.csv')
write_out_csv(blast_probe_file.abspath, probe_blasts, append=False)
"""Convert Blast list into field:val dict for db import"""
log.name = ('Probe:BlastListtPrepImport')
log.info('Converting list of blast hits to dict for import to db')
probe_fields = probe_blasts.pop(0) # pop off blast_header record with new columns
pseqs = []
if len(probe_fields) == len(probe_blasts[0]):
for vals in probe_blasts:
vals_dict = {f:v for (f,v) in zip(probe_fields, vals)}
pseqs.append(vals_dict)
log.debug(f'len pseqs: {len(pseqs)}')
"""import blast file to cluster database"""
log.name = 'Probe:ImportBlast'
db_name = DB_CFG.get('clusterdb').get('name')
clust_db = working_dir / '_'.join([cluster_id, db_name])
log.info(f'Importing blast matches to db "{clust_db}"')
import_blasts_to_db(pseqs, db_name=clust_db.abspath)
"""Filter resulting table to limits in CONFIG"""
log.name = 'Probe:FilterView'
filter_probe_seqs(clust_db.abspath, cluster_id)
"""Create two views, one for SC, one inverse for MC"""
log.name = 'Probe:ExportFinals'
final_probe_amount = CONFIG.get('general').get('final_probe_amount')
log.debug(f' ... final_probe_amount {final_probe_amount}')
export_final_sets(clust_db.abspath, cluster_id, final_probe_amount=final_probe_amount)
else:
log.notice(f'Number of fieldnames({len(probe_fields)}) not equal to'
f'number of values({len(probe_blasts[0])})!')
return probes_file