args = argparser.parse_args()
alignments = pysam.AlignmentFile(args.alignmentfile)
if args.bamout is not None:
temp_out = f'{args.bamout}.unsorted.bam'
output = pysam.AlignmentFile(temp_out, "wb", header=alignments.header)
else:
output = None
samples = None if args.samples is None else set(args.samples.split(','))
contexts = None if args.context is None else set(
[x.upper() for x in args.context.split(',')] +
[x.lower() for x in args.context.split(',')])
if args.ref is None:
args.ref = bf.get_reference_from_pysam_alignmentFile(alignments)
if args.ref is None:
raise ValueError("Supply reference, -ref")
reference = pysamiterators.iterators.CachedFasta(pysam.FastaFile(args.ref))
taps = singlecellmultiomics.molecule.TAPS(reference=reference)
molecule_class_args = {
'reference': reference,
'taps': taps,
'min_max_mapping_quality': args.minmq
}
if args.method == 'nla':
moleculeClass = singlecellmultiomics.molecule.TAPSNlaIIIMolecule
fragmentClass = singlecellmultiomics.fragment.NLAIIIFragment
molecule_class_args.update({'site_has_to_be_mapped': True})
def run_multiome_tagging(args):
"""
Run multiome tagging adds molecule information
Arguments:
bamin (str) : bam file to process
o(str) : path to output bam file
method(str): Protocol to tag, select from:nla, qflag, chic, nla_transcriptome, vasa, cs, nla_taps ,chic_taps, nla_no_overhang, scartrace
qflagger(str): Query flagging algorithm to use, this algorithm extracts UMI and sample information from your reads. When no query flagging algorithm is specified, the `singlecellmultiomics.universalBamTagger.universalBamTagger.QueryNameFlagger` is used
method(str) : Method name, what kind of molecules need to be extracted. Select from:
nla (Data with digested by Nla III enzyme)
qflag (Only add basic tags like sampple and UMI, no molecule assignment)
chic (Data digested using mnase fusion)
nla_transcriptome (Data with transcriptome and genome digested by Nla III )
vasa (VASA transcriptomic data)
cs (CELseq data, 1 and 2)
cs_feature_counts (Single end, deduplicate using a bam file tagged using featurecounts, deduplicates a umi per gene)
fl_feature_counts (deduplicate using a bam file tagged using featurecounts, deduplicates based on fragment position)
nla_taps (Data with digested by Nla III enzyme and methylation converted by TAPS)
chic_taps (Data with digested by mnase enzyme and methylation converted by TAPS)
chic_nla
scartrace (lineage tracing protocol)
custom_flags(str): Arguments passed to the query name flagger, comma separated "MI,RX,bi,SM"
ref(str) : Path to reference fasta file, autodected from bam header when not supplied
umi_hamming_distance(int) : Max hamming distance on UMI's
head (int) : Amount of molecules to process
contig (str) : only process this contig
region_start(int) : Zero based start coordinate of single region to process
region_end(int) : Zero based end coordinate of single region to process, None: all contigs when contig is not set, complete contig when contig is set.
alleles (str) : path to allele VCF
allele_samples(str): Comma separated samples to extract from the VCF file. For example B6,SPRET
unphased_alleles(str) : Path to VCF containing unphased germline SNPs
mapfile (str) : 'Path to \*.safe.bgzf file, used to decide if molecules are uniquely mappable, generate one using createMapabilityIndex.py
annotmethod (int) : Annotation resolving method. 0: molecule consensus aligned blocks. 1: per read per aligned base
cluster (bool) : Run contigs in separate cluster jobs
resolve_unproperly_paired_reads(bool) : When enabled bamtagmultiome will look through the complete bam file in a hunt for the mate, the two mates will always end up in 1 molecule if both present in the bam file. This also works when the is_proper_pair bit is not set. Use this option when you want to find the breakpoints of genomic re-arrangements.
no_rejects(bool) : Do not write rejected reads
mem (int) : Amount of gigabytes to request for cluster jobs
time(int) : amount of wall clock hours to request for cluster jobs
exons(str): Path to exon annotation GTF file
introns(str): Path to intron annotation GTF file
consensus(bool) : Calculate molecule consensus read, this feature is _VERY_ experimental
consensus_model(str) : Path to consensus calling model, when none specified, this is learned based on the supplied bam file, ignoring sites supplied by -consensus_mask_variants
consensus_mask_variants(str): Path VCF file masked for training on consensus caller
consensus_n_train(int) : Amount of bases used for training the consensus model
no_source_reads(bool) : Do not write original reads, only consensus
scartrace_r1_primers(str) : comma separated list of R1 primers used in scartrace protocol
"""
MISC_ALT_CONTIGS_SCMO = 'MISC_ALT_CONTIGS_SCMO'
every_fragment_as_molecule = args.every_fragment_as_molecule
skip_contig = set(args.skip_contig.split(',')) if args.skip_contig is not None else set()
if not args.o.endswith('.bam'):
raise ValueError(
"Supply an output which ends in .bam, for example -o output.bam")
write_status(args.o,'unfinished')
# Verify wether the input file is indexed and sorted...
if not args.ignore_bam_issues:
verify_and_fix_bam(args.bamin)
for remove_existing_path in [args.o, f'{args.o}.bai']:
if os.path.exists(remove_existing_path):
print(f"Removing existing file {remove_existing_path}")
os.remove(remove_existing_path)
input_bam = pysam.AlignmentFile(args.bamin, "rb", ignore_truncation=args.ignore_bam_issues, threads=4)
# autodetect reference:
reference = None
if args.ref is None:
args.ref = get_reference_from_pysam_alignmentFile(input_bam)
if args.ref is not None:
try:
reference = CachedFasta(
pysam.FastaFile(args.ref))
print(f'Loaded reference from {args.ref}')
except Exception as e:
print("Error when loading the reference file, continuing without a reference")
reference = None
##### Define fragment and molecule class arguments and instances: ####
queryNameFlagger = None
if args.qflagger is not None:
if args.qflagger == 'custom_flags':
queryNameFlagger = CustomAssingmentQueryNameFlagger(
args.custom_flags.split(','))
else:
raise ValueError("Select from 'custom_flags, ..' ")
molecule_class_args = {
'umi_hamming_distance': args.umi_hamming_distance,
'reference': reference
}
fragment_class_args = {
'read_group_format' : args.read_group_format
}
yield_invalid = True # if invalid reads should be written
yield_overflow = True # if overflow reads should be written
if args.max_fragment_size is not None:
fragment_class_args['max_fragment_size'] = args.max_fragment_size
if args.no_rejects:
yield_invalid = False
if args.no_overflow:
yield_overflow = False
ignore_conversions = None
if args.method == 'nla_taps' or args.method == 'chic_taps':
ignore_conversions = set([('C', 'T'), ('G', 'A')])
if args.alleles is not None:
molecule_class_args['allele_resolver'] = singlecellmultiomics.alleleTools.AlleleResolver(
args.alleles,
select_samples=args.allele_samples.split(',') if args.allele_samples is not None else None,
lazyLoad=True,
use_cache=args.use_allele_cache,
verbose = args.set_allele_resolver_verbose,
ignore_conversions=ignore_conversions)
if args.mapfile is not None:
molecule_class_args['mapability_reader'] = MapabilityReader(
args.mapfile)
### Transcriptome configuration ###
if args.method in ('nla_transcriptome', 'cs', 'vasa'):
print(
colorama.Style.BRIGHT +
'Running in transcriptome annotation mode' +
colorama.Style.RESET_ALL)
if args.exons is None :
raise ValueError("Supply an exon GTF file")
if args.introns is not None and args.exons is None:
raise ValueError("Please supply both intron and exon GTF files")
transcriptome_features = singlecellmultiomics.features.FeatureContainer()
print("Loading exons", end='\r')
transcriptome_features.loadGTF(
args.exons,
select_feature_type=['exon'],
identifierFields=(
'exon_id',
'gene_id'),
store_all=True,
contig=args.contig,
head=None)
if args.introns is not None:
print("Loading introns", end='\r')
transcriptome_features.loadGTF(
args.introns,
select_feature_type=['intron'],
identifierFields=['transcript_id'],
store_all=True,
contig=args.contig,
head=None)
print("All features loaded")
# Add more molecule class arguments
molecule_class_args.update({
'features': transcriptome_features,
'auto_set_intron_exon_features': True
})
### Method specific configuration ###
if args.method == 'qflag':
moleculeClass = singlecellmultiomics.molecule.Molecule
fragmentClass = singlecellmultiomics.fragment.Fragment
# Write all reads
yield_invalid = True
elif args.method == 'chic':
moleculeClass = singlecellmultiomics.molecule.CHICMolecule
fragmentClass = singlecellmultiomics.fragment.CHICFragment
elif args.method == 'nla' or args.method == 'nla_no_overhang':
moleculeClass = singlecellmultiomics.molecule.NlaIIIMolecule
fragmentClass = singlecellmultiomics.fragment.NLAIIIFragment
if args.method == 'nla_no_overhang':
assert reference is not None, 'Supply a reference fasta using -ref!'
fragment_class_args.update({
'reference': reference,
'no_overhang': True
})
elif args.method == 'chic_nla':
moleculeClass=singlecellmultiomics.molecule.CHICNLAMolecule
fragmentClass=singlecellmultiomics.fragment.CHICFragment
assert reference is not None, 'Supply a reference fasta using -ref!'
molecule_class_args.update({
'reference': reference,
})
elif args.method == 'cs_feature_counts' :
moleculeClass = singlecellmultiomics.molecule.Molecule
fragmentClass = singlecellmultiomics.fragment.FeatureCountsSingleEndFragment
elif args.method == 'fl_feature_counts':
moleculeClass = singlecellmultiomics.molecule.Molecule
fragmentClass = singlecellmultiomics.fragment.FeatureCountsFullLengthFragment
elif args.method == 'episeq' :
moleculeClass = singlecellmultiomics.molecule.Molecule
fragmentClass = singlecellmultiomics.fragment.FeatureCountsSingleEndFragment
elif args.method == 'nla_transcriptome':
moleculeClass = singlecellmultiomics.molecule.AnnotatedNLAIIIMolecule
fragmentClass = singlecellmultiomics.fragment.NLAIIIFragment
molecule_class_args.update({
'pooling_method': 1, # all data from the same cell can be dealt with separately
'stranded': None # data is not stranded
})
elif args.method == 'nla_taps':
moleculeClass = singlecellmultiomics.molecule.TAPSNlaIIIMolecule
fragmentClass = singlecellmultiomics.fragment.NLAIIIFragment
molecule_class_args.update({
'reference': reference,
'taps': singlecellmultiomics.molecule.TAPS(reference=reference)
})
elif args.method == 'chic_taps':
molecule_class_args.update({
'reference': reference,
'taps': singlecellmultiomics.molecule.TAPS(reference=reference)
})
moleculeClass = singlecellmultiomics.molecule.TAPSCHICMolecule
fragmentClass = singlecellmultiomics.fragment.CHICFragment
elif args.method == 'vasa' or args.method == 'cs':
moleculeClass = singlecellmultiomics.molecule.VASA
fragmentClass = singlecellmultiomics.fragment.SingleEndTranscript
molecule_class_args.update({
'pooling_method': 1, # all data from the same cell can be dealt with separately
'stranded': 1 # data is stranded
})
elif args.method == 'scartrace':
moleculeClass = singlecellmultiomics.molecule.ScarTraceMolecule
fragmentClass = singlecellmultiomics.fragment.ScarTraceFragment
r1_primers = args.scartrace_r1_primers.split(',')
fragment_class_args.update({
'scartrace_r1_primers': r1_primers,
#'reference': reference
})
else:
raise ValueError("Supply a valid method")
# Allow or disallow cycle shift:
if args.allow_cycle_shift and fragmentClass is singlecellmultiomics.fragment.NLAIIIFragment:
fragment_class_args['allow_cycle_shift'] = True
# This disables umi_cigar_processing:
if args.no_umi_cigar_processing:
fragment_class_args['no_umi_cigar_processing'] = True
if args.max_associated_fragments is not None:
molecule_class_args['max_associated_fragments'] = args.max_associated_fragments
# This decides what molecules we will traverse
if args.contig == MISC_ALT_CONTIGS_SCMO:
contig = None
else:
contig = args.contig
# This decides to only extract a single genomic region:
if args.region_start is not None:
if args.region_end is None:
raise ValueError('When supplying -region_start then also supply -region_end')
region_start = args.region_start
region_end = args.region_end
else:
region_start = None
region_end = None
last_update = datetime.now()
init_time = datetime.now()
if args.molecule_iterator_verbosity_interval is not None and (args.molecule_iterator_verbose or (args.stats_file_path is not None )):
stats_handle = None
if args.stats_file_path is not None:
stats_handle = open(args.stats_file_path,'w')
def progress_callback_function( iteration, mol_iter, reads ):
nonlocal last_update
nonlocal init_time
nonlocal stats_handle
now = datetime.now()
diff = (datetime.now()-last_update).total_seconds()
if diff>args.molecule_iterator_verbosity_interval:
diff_from_init = (datetime.now()-init_time).total_seconds()
_contig, _pos = None, None
for read in reads:
if read is not None:
_contig, _pos = read.reference_name, read.reference_start
if args.molecule_iterator_verbose:
print( f'{mol_iter.yielded_fragments} fragments written, {mol_iter.deleted_fragments} fragments deleted ({(mol_iter.deleted_fragments/(mol_iter.deleted_fragments + mol_iter.yielded_fragments))*100:.2f} %), current pos: {_contig}, {_pos}, {mol_iter.waiting_fragments} fragments waiting ' , end='\r')
if stats_handle is not None:
stats_handle.write(f'{diff_from_init}\t{mol_iter.waiting_fragments}\t{mol_iter.yielded_fragments}\t{mol_iter.deleted_fragments}\t{_contig}\t{_pos}\n')
stats_handle.flush()
last_update = now
else:
progress_callback_function = None
molecule_iterator_args = {
'alignments': input_bam,
'queryNameFlagger': queryNameFlagger,
'moleculeClass': moleculeClass,
'fragmentClass': fragmentClass,
'molecule_class_args': molecule_class_args,
'fragment_class_args': fragment_class_args,
'yield_invalid': yield_invalid,
'yield_overflow': yield_overflow,
'start':region_start,
'end':region_end,
'contig': contig,
'every_fragment_as_molecule': every_fragment_as_molecule,
'skip_contigs':skip_contig,
'progress_callback_function':progress_callback_function
}
if args.resolve_unproperly_paired_reads:
molecule_iterator_args['iterator_class'] = MatePairIteratorIncludingNonProper
if args.contig == MISC_ALT_CONTIGS_SCMO:
# When MISC_ALT_CONTIGS_SCMO is set as argument, all molecules with reads
# mapping to a contig returning True from the is_main_chromosome
# function are used
def Misc_contig_molecule_generator(molecule_iterator_args):
for reference in input_bam.references:
if not is_main_chromosome(reference):
molecule_iterator_args['contig'] = reference
yield from MoleculeIterator(**molecule_iterator_args)
molecule_iterator = Misc_contig_molecule_generator(
molecule_iterator_args)
else:
molecule_iterator = MoleculeIterator(**molecule_iterator_args)
#####
consensus_model_path = None
if args.consensus:
# Load from path if available:
if args.consensus_model is not None:
if os.path.exists(args.consensus_model):
model_path = args.consensus_model
else:
model_path = pkg_resources.resource_filename(
'singlecellmultiomics', f'molecule/consensus_model/{args.consensus_model}')
if model_path.endswith('.h5'):
try:
from tensorflow.keras.models import load_model
except ImportError:
print("Please install tensorflow")
raise
consensus_model = load_model(model_path)
else:
with open(model_path, 'rb') as f:
consensus_model = pickle.load(f)
else:
skip_already_covered_bases = not args.consensus_allow_train_location_oversampling
if args.consensus_mask_variants is None:
mask_variants = None
else:
mask_variants = pysam.VariantFile(args.consensus_mask_variants)
print("Fitting consensus model, this may take a long time")
consensus_model = singlecellmultiomics.molecule.train_consensus_model(
molecule_iterator,
mask_variants=mask_variants,
n_train=args.consensus_n_train,
skip_already_covered_bases=skip_already_covered_bases
)
# Write the consensus model to disk
consensus_model_path = os.path.abspath(
os.path.dirname(args.o)) + '/consensus_model.pickle.gz'
print(f'Writing consensus model to {consensus_model_path}')
with open(consensus_model_path, 'wb') as f:
pickle.dump(consensus_model, f)
# We needed to check if every argument is properly placed. If so; the jobs
# can be sent to the cluster
if args.cluster:
if args.contig is None:
write_status(args.o,'Submitting jobs. If this file remains, a job failed.')
# Create jobs for all chromosomes:
unique_id = str(uuid.uuid4())
temp_prefix = os.path.abspath(os.path.dirname(
args.o)) + '/SCMO_' + unique_id
hold_merge = []
## Create folder to store cluster files:
if args.clusterdir is None:
cluster_file_folder = os.path.abspath(os.path.dirname(
args.o)) + '/cluster'
else:
cluster_file_folder = args.clusterdir
print(f'Writing cluster scripts and standard out and error to {cluster_file_folder}')
if not os.path.exists(cluster_file_folder):
try:
os.makedirs(cluster_file_folder,exist_ok=True)
except Exception as e:
print(e)
pass
found_alts = 0
files_to_merge = []
for ci,chrom in enumerate([_chrom for _chrom in
(list(input_bam.references) + [MISC_ALT_CONTIGS_SCMO])
if not _chrom in skip_contig]):
if not is_main_chromosome(chrom):
found_alts += 1
continue
if chrom == MISC_ALT_CONTIGS_SCMO and found_alts == 0:
continue
temp_bam_path = f'{temp_prefix}_{chrom}.bam'
if os.path.exists(temp_bam_path):
print(f"Removing existing temporary file {temp_bam_path}")
os.remove(temp_bam_path)
arguments = " ".join(
[x for x in sys.argv if not x == args.o and x != '-o']) + f" -contig {chrom} -o {temp_bam_path}"
files_to_merge.append(temp_bam_path)
if consensus_model_path is not None:
arguments += f' -consensus_model {consensus_model_path}'
job = f'SCMULTIOMICS_{ci}_{unique_id}'
write_status(temp_bam_path,'SUBMITTED')
job_id = submit_job(f'{arguments};', job_name=job, target_directory=cluster_file_folder, working_directory=None,
threads_n=1, memory_gb=args.mem, time_h=args.time, scheduler=args.sched, copy_env=True,
email=None, mail_when_finished=False, hold=None,submit=True)
print(f'Job for contig {chrom} submitted with job id: {job_id}')
hold_merge.append(job_id)
hold = hold_merge
job = f'SCMULTIOMICS_MERGE_{unique_id}'
if args.sched == 'local':
hold = None
final_status = args.o.replace('.bam','.status.txt')
# Create list of output files
command = f'samtools merge -@ 4 -c {args.o} {" ".join(files_to_merge)} && samtools index {args.o} && rm {temp_prefix}*.ba* && rm {temp_prefix}*.status.txt && echo "All done" > {final_status}'
final_job_id = submit_job(f'{command};', job_name=job, target_directory=cluster_file_folder, working_directory=None,
threads_n=4, memory_gb=10, time_h=args.time, scheduler=args.sched, copy_env=True,
email=None, mail_when_finished=False, hold=hold,submit=True)
print(f'final job id is:{final_job_id}')
exit()
#####
# Load unphased variants to memory
unphased_allele_resolver = None
if args.unphased_alleles is not None:
unphased_allele_resolver = singlecellmultiomics.alleleTools.AlleleResolver(
use_cache=args.use_allele_cache,
phased=False, ignore_conversions=ignore_conversions,verbose = args.set_allele_resolver_verbose)
try:
for i, variant in enumerate(
pysam.VariantFile(
args.unphased_alleles).fetch(
args.contig)):
if 'PASS' not in list(variant.filter):
continue
if not all(
len(allele) == 1 for allele in variant.alleles) or len(
variant.alleles) != 2:
continue
if sum([len(set(variant.samples[sample].alleles))
== 2 for sample in variant.samples]) < 2:
# Not heterozygous
continue
unphased_allele_resolver.locationToAllele[variant.chrom][variant.pos - 1] = {
variant.alleles[0]: {'U'}, variant.alleles[1]: {'V'}}
except Exception as e: # todo catch this more nicely
print(e)
out_bam_path = args.o
# Copy the header
input_header = input_bam.header.as_dict()
# Write provenance information to BAM header
write_program_tag(
input_header,
program_name='bamtagmultiome',
command_line=" ".join(
sys.argv),
version=singlecellmultiomics.__version__,
description=f'SingleCellMultiOmics molecule processing, executed at {datetime.now().strftime("%d/%m/%Y %H:%M:%S")}')
print(f'Started writing to {out_bam_path}')
read_groups = dict() # Store unique read groups in this dict
with sorted_bam_file(out_bam_path, header=input_header, read_groups=read_groups) as out:
try:
for i, molecule in enumerate(molecule_iterator):
# Stop when enough molecules are processed
if args.head is not None and (i - 1) >= args.head:
break
# set unique molecule identifier
molecule.set_meta('mi', f'{molecule.get_a_reference_id()}_{i}')
# Write tag values
molecule.write_tags()
if unphased_allele_resolver is not None: # write unphased allele tag:
molecule.write_allele_phasing_information_tag(
unphased_allele_resolver, 'ua')
# Update read groups
for fragment in molecule:
rgid = fragment.get_read_group()
if not rgid in read_groups:
read_groups[rgid] = fragment.get_read_group(True)[1]
# Calculate molecule consensus
if args.consensus:
try:
consensus_reads = molecule.deduplicate_to_single_CIGAR_spaced(
out,
f'consensus_{molecule.get_a_reference_id()}_{i}',
consensus_model,
NUC_RADIUS=args.consensus_k_rad
)
for consensus_read in consensus_reads:
consensus_read.set_tag('RG', molecule[0].get_read_group())
consensus_read.set_tag('mi', i)
out.write(consensus_read)
except Exception as e:
#traceback.print_exc()
#print(e)
molecule.set_rejection_reason('CONSENSUS_FAILED',set_qcfail=True)
molecule.write_pysam(out)
# Write the reads to the output file
if not args.no_source_reads:
molecule.write_pysam(out)
except Exception as e:
write_status(args.o,'FAIL, The file is not complete')
raise e
# Reached the end of the generator
write_status(args.o,'Reached end. All ok!')