def run_haplotag(
variant_file,
alignment_file,
output=None,
reference=None,
regions=None,
ignore_linked_read=False,
given_samples=None,
linked_read_distance_cutoff=50000,
ignore_read_groups=False,
haplotag_list=None,
tag_supplementary=False,
):
timers = StageTimer()
timers.start("haplotag-run")
with ExitStack() as stack:
timers.start("haplotag-init")
try:
vcf_reader = stack.enter_context(
VcfReader(variant_file, indels=True, phases=True))
except OSError as err:
logger.error("Error while loading variant file {}: {}".format(
variant_file, err))
raise err
use_vcf_samples = compute_variant_file_samples_to_use(
vcf_reader, given_samples, ignore_read_groups)
try:
bam_reader = stack.enter_context(
pysam.AlignmentFile(alignment_file, "rb", require_index=True))
except OSError as err:
logger.error("Error while loading alignment file {}: {}".format(
alignment_file, err))
raise err
# This checks also sample compatibility with VCF
shared_samples = compute_shared_samples(bam_reader, ignore_read_groups,
use_vcf_samples)
# Check if user has specified a subset of regions per chromosome
user_regions = normalize_user_regions(regions, bam_reader.references)
phased_input_reader = stack.enter_context(
PhasedInputReader([alignment_file],
reference,
NumericSampleIds(),
ignore_read_groups,
indels=False))
bam_writer = stack.enter_context(
open_output_alignment_file(
output,
reference,
md5_of(variant_file),
bam_reader.header.to_dict(),
))
haplotag_writer = stack.enter_context(
open_haplotag_writer(haplotag_list))
timers.stop("haplotag-init")
logger.debug("All input/output files initialized (time: {})".format(
timers.elapsed("haplotag-init")))
timers.start("haplotag-process")
n_alignments = 0
n_tagged = 0
n_multiple_phase_sets = 0
for chrom, regions in user_regions.items():
logger.debug("Processing chromosome {}".format(chrom))
variant_table = load_chromosome_variants(vcf_reader, chrom,
regions)
if variant_table is not None:
logger.debug("Preparing haplotype information")
(BX_tag_to_haplotype, read_to_haplotype,
n_mult) = prepare_haplotag_information(
variant_table,
shared_samples,
phased_input_reader,
regions,
ignore_linked_read,
linked_read_distance_cutoff,
)
n_multiple_phase_sets += n_mult
else:
# avoid uninitialized variables
BX_tag_to_haplotype = None
read_to_haplotype = None
for start, end in regions:
logger.debug("Iterating chromosome regions")
for alignment in bam_reader.fetch(contig=chrom,
start=start,
stop=end):
n_alignments += 1
haplotype_name = "none"
phaseset = "none"
alignment.set_tag("HP", value=None)
alignment.set_tag("PC", value=None)
alignment.set_tag("PS", value=None)
if variant_table is None or ignore_read(
alignment, tag_supplementary):
# - If no variants in VCF for this chromosome,
# alignments just get written to output
# - Ignored reads are simply
# written to the output BAM
pass
else:
(is_tagged, haplotype_name,
phaseset) = attempt_add_phase_information(
alignment,
read_to_haplotype,
BX_tag_to_haplotype,
linked_read_distance_cutoff,
)
n_tagged += is_tagged
bam_writer.write(alignment)
if not (alignment.is_secondary
or alignment.is_supplementary):
print(
alignment.query_name,
haplotype_name,
phaseset,
chrom,
sep="\t",
file=haplotag_writer,
)
if n_alignments % 100000 == 0:
logger.debug("Processed {} alignment records.".format(
n_alignments))
timers.stop("haplotag-process")
logger.debug("Processing complete (time: {})".format(
timers.elapsed("haplotag-process")))
timers.stop("haplotag-run")
logger.info("\n== SUMMARY ==")
logger.info("Total alignments processed: %12d", n_alignments)
logger.info("Alignments that could be tagged: %12d", n_tagged)
logger.info("Alignments spanning multiple phase sets: %12d",
n_multiple_phase_sets)
logger.info("haplotag - total processing time: {}".format(
timers.elapsed("haplotag-run")))
def run_whatshap(
phase_input_files: List[str],
variant_file: str,
reference: Union[None, bool, str] = False,
output: TextIO = sys.stdout,
samples: List[str] = None,
chromosomes: Optional[List[str]] = None,
ignore_read_groups: bool = False,
indels: bool = True,
mapping_quality: int = 20,
read_merging: bool = False,
read_merging_error_rate: float = 0.15,
read_merging_max_error_rate: float = 0.25,
read_merging_positive_threshold: int = 1000000,
read_merging_negative_threshold: int = 1000,
max_coverage: int = 15,
distrust_genotypes: bool = False,
include_homozygous: bool = False,
ped: Optional[str] = None,
recombrate: float = 1.26,
genmap: Optional[str] = None,
genetic_haplotyping: bool = True,
recombination_list_filename: Optional[str] = None,
tag: str = "PS",
read_list_filename: Optional[str] = None,
gl_regularizer: Optional[float] = None,
gtchange_list_filename: Optional[str] = None,
default_gq: int = 30,
write_command_line_header: bool = True,
use_ped_samples: bool = False,
algorithm: str = "whatshap",
):
"""
Run WhatsHap.
phase_input_files -- list of paths to BAM/CRAM/VCF files
variant_file -- path to input VCF
reference -- path to reference FASTA. If False: skip realignment. If None: complain if reference needed.
output -- path to output VCF or a file-like object
samples -- names of samples to phase. an empty list means: phase all samples
chromosomes -- names of chromosomes to phase. an empty list means: phase all chromosomes
ignore_read_groups
mapping_quality -- discard reads below this mapping quality
read_merging -- whether or not to merge reads
read_merging_error_rate -- probability that a nucleotide is wrong
read_merging_max_error_rate -- max error rate on edge of merge graph considered
read_merging_positive_threshold -- threshold on the ratio of the two probabilities
read_merging_negative_threshold -- threshold on the opposite ratio of positive threshold
max_coverage
distrust_genotypes
include_homozygous
genetic_haplotyping -- in ped mode, merge disconnected blocks based on genotype status
recombination_list_filename -- filename to write putative recombination events to
tag -- How to store phasing info in the VCF, can be 'PS' or 'HP'
read_list_filename -- name of file to write list of used reads to
algorithm -- algorithm to use, can be 'whatshap' or 'hapchat'
gl_regularizer -- float to be passed as regularization constant to GenotypeLikelihoods.as_phred
gtchange_list_filename -- filename to write list of changed genotypes to
default_gq -- genotype likelihood to be used when GL or PL not available
write_command_line_header -- whether to add a ##commandline header to the output VCF
"""
if algorithm == "hapchat" and ped is not None:
raise CommandLineError(
"The hapchat algorithm cannot do pedigree phasing")
timers = StageTimer()
logger.info(
f"This is WhatsHap {__version__} running under Python {platform.python_version()}"
)
numeric_sample_ids = NumericSampleIds()
command_line: Optional[str]
if write_command_line_header:
command_line = "(whatshap {}) {}".format(__version__,
" ".join(sys.argv[1:]))
else:
command_line = None
read_merger: ReadMergerBase
if read_merging:
read_merger = ReadMerger(
read_merging_error_rate,
read_merging_max_error_rate,
read_merging_positive_threshold,
read_merging_negative_threshold,
)
else:
read_merger = DoNothingReadMerger()
with ExitStack() as stack:
try:
vcf_writer = stack.enter_context(
PhasedVcfWriter(
command_line=command_line,
in_path=variant_file,
out_file=output,
tag=tag,
indels=indels,
))
except (OSError, VcfError) as e:
raise CommandLineError(e)
phased_input_reader = stack.enter_context(
PhasedInputReader(
phase_input_files,
None if reference is False else reference,
numeric_sample_ids,
ignore_read_groups,
mapq_threshold=mapping_quality,
indels=indels,
))
show_phase_vcfs = phased_input_reader.has_vcfs
if phased_input_reader.has_alignments and reference is None:
raise CommandLineError(
"A reference FASTA needs to be provided with -r/--reference; "
"or use --no-reference at the expense of phasing quality.")
# Only read genotype likelihoods from VCFs when distrusting genotypes
vcf_reader = stack.enter_context(
VcfReader(variant_file,
indels=indels,
genotype_likelihoods=distrust_genotypes))
if ignore_read_groups and not samples and len(vcf_reader.samples) > 1:
raise CommandLineError(
"When using --ignore-read-groups on a VCF with "
"multiple samples, --sample must also be used.")
if not samples:
samples = vcf_reader.samples
# if --use-ped-samples is set, use only samples from PED file
if ped and use_ped_samples:
samples = PedReader(ped).samples()
raise_if_any_sample_not_in_vcf(vcf_reader, samples)
recombination_cost_computer = make_recombination_cost_computer(
ped, genmap, recombrate)
families, family_trios = setup_families(samples, ped, max_coverage)
del samples
for trios in family_trios.values():
for trio in trios:
# Ensure that all mentioned individuals have a numeric id
_ = numeric_sample_ids[trio.child]
read_list = None
if read_list_filename:
read_list = stack.enter_context(ReadList(read_list_filename))
if algorithm == "hapchat":
logger.warning(
"On which haplotype a read occurs in the inferred solution is not yet "
"implemented in hapchat, and so the corresponding column in the "
"read list file contains no information about this")
with timers("parse_phasing_vcfs"):
# TODO should this be done in PhasedInputReader.__init__?
phased_input_reader.read_vcfs()
superreads: Dict[str, ReadSet]
components: Dict
for variant_table in timers.iterate("parse_vcf", vcf_reader):
chromosome = variant_table.chromosome
if (not chromosomes) or (chromosome in chromosomes):
logger.info("======== Working on chromosome %r", chromosome)
else:
logger.info(
"Leaving chromosome %r unchanged (present in VCF but not requested by option --chromosome)",
chromosome,
)
with timers("write_vcf"):
superreads, components = dict(), dict()
vcf_writer.write(chromosome, superreads, components)
continue
# These two variables hold the phasing results for all samples
superreads, components = dict(), dict()
# Iterate over all families to process, i.e. a separate DP table is created
# for each family.
# TODO: Can the body of this loop be factored out into a phase_family function?
for representative_sample, family in sorted(families.items()):
if len(family) == 1:
logger.info("---- Processing individual %s",
representative_sample)
else:
logger.info("---- Processing family with individuals: %s",
",".join(family))
max_coverage_per_sample = max(1, max_coverage // len(family))
logger.info("Using maximum coverage per sample of %dX",
max_coverage_per_sample)
trios = family_trios[representative_sample]
assert len(family) == 1 or len(trios) > 0
homozygous_positions, phasable_variant_table = find_phaseable_variants(
family, include_homozygous, trios, variant_table)
# Get the reads belonging to each sample
readsets = dict() # TODO this could become a list
for sample in family:
with timers("read_bam"):
readset, vcf_source_ids = phased_input_reader.read(
chromosome, phasable_variant_table.variants,
sample)
# TODO: Read selection done w.r.t. all variants, where using heterozygous
# variants only would probably give better results.
with timers("select"):
readset = readset.subset([
i for i, read in enumerate(readset)
if len(read) >= 2
])
logger.info(
"Kept %d reads that cover at least two variants each",
len(readset))
merged_reads = read_merger.merge(readset)
selected_reads = select_reads(
merged_reads,
max_coverage_per_sample,
preferred_source_ids=vcf_source_ids,
)
readsets[sample] = selected_reads
if len(family) == 1 and not distrust_genotypes:
# When having a pedigree (len(family) > 1), blocks are also merged after
# phasing based on the pedigree information and these statistics are not
# so useful. When distrust_genotypes, genotypes can change during phasing
# and so can the block structure. So don't print these stats in those cases
log_best_case_phasing_info(readset, selected_reads)
all_reads = merge_readsets(readsets)
# Determine which variants can (in principle) be phased
accessible_positions = sorted(all_reads.get_positions())
logger.info(
"Variants covered by at least one phase-informative "
"read in at least one individual after read selection: %d",
len(accessible_positions),
)
if len(family) > 1 and genetic_haplotyping:
# In case of genetic haplotyping, also retain all positions homozygous
# in at least one individual (because they might be phased based on genotypes)
accessible_positions = sorted(
set(accessible_positions).union(homozygous_positions))
logger.info(
"Variants either covered by phase-informative read or homozygous "
"in at least one individual: %d",
len(accessible_positions),
)
# Keep only accessible positions
phasable_variant_table.subset_rows_by_position(
accessible_positions)
assert len(phasable_variant_table.variants) == len(
accessible_positions)
pedigree = create_pedigree(
default_gq,
distrust_genotypes,
family,
gl_regularizer,
numeric_sample_ids,
phasable_variant_table,
trios,
)
recombination_costs = recombination_cost_computer.compute(
accessible_positions)
# Finally, run phasing algorithm
with timers("phase"):
problem_name = "MEC" if len(family) == 1 else "PedMEC"
logger.info(
"Phasing %d sample%s by solving the %s problem ...",
len(family),
plural_s(len(family)),
problem_name,
)
dp_table: Union[HapChatCore, PedigreeDPTable]
if algorithm == "hapchat":
dp_table = HapChatCore(all_reads)
else:
dp_table = PedigreeDPTable(
all_reads,
recombination_costs,
pedigree,
distrust_genotypes,
accessible_positions,
)
superreads_list, transmission_vector = dp_table.get_super_reads(
)
logger.info("%s cost: %d", problem_name,
dp_table.get_optimal_cost())
with timers("components"):
overall_components = compute_overall_components(
accessible_positions,
all_reads,
distrust_genotypes,
family,
genetic_haplotyping,
homozygous_positions,
numeric_sample_ids,
superreads_list,
)
log_component_stats(overall_components,
len(accessible_positions))
if recombination_list_filename:
n_recombinations = write_recombination_list(
recombination_list_filename,
chromosome,
accessible_positions,
overall_components,
recombination_costs,
transmission_vector,
trios,
)
logger.info(
"Total no. of detected recombination events: %d",
n_recombinations)
# Superreads in superreads_list are in the same order as individuals were added to the pedigree
for sample, sample_superreads in zip(family, superreads_list):
superreads[sample] = sample_superreads
assert len(sample_superreads) == 2
assert (sample_superreads[0].sample_id ==
sample_superreads[1].sample_id ==
numeric_sample_ids[sample])
# identical for all samples
components[sample] = overall_components
if read_list:
read_list.write(
all_reads,
dp_table.get_optimal_partitioning(),
components,
numeric_sample_ids,
)
with timers("write_vcf"):
logger.info("======== Writing VCF")
changed_genotypes = vcf_writer.write(chromosome, superreads,
components)
logger.info("Done writing VCF")
if changed_genotypes:
assert distrust_genotypes
logger.info("Changed %d genotypes while writing VCF",
len(changed_genotypes))
if gtchange_list_filename:
logger.info("Writing list of changed genotypes to %r",
gtchange_list_filename)
write_changed_genotypes(gtchange_list_filename,
changed_genotypes)
logger.debug("Chromosome %r finished", chromosome)
log_time_and_memory_usage(timers, show_phase_vcfs=show_phase_vcfs)
def run_genotype(
phase_input_files,
variant_file,
reference=None,
output=sys.stdout,
samples=None,
chromosomes=None,
ignore_read_groups=False,
indels=True,
mapping_quality=20,
max_coverage=15,
nopriors=False,
ped=None,
recombrate=1.26,
genmap=None,
gt_qual_threshold=0,
prioroutput=None,
constant=0.0,
overhang=10,
affine_gap=False,
gap_start=10,
gap_extend=7,
mismatch=15,
write_command_line_header=True,
use_ped_samples=False,
):
"""
For now: this function only runs the genotyping algorithm. Genotype likelihoods for
all variants are computed using the forward backward algorithm
"""
timers = StageTimer()
logger.info(
"This is WhatsHap (genotyping) %s running under Python %s",
__version__,
platform.python_version(),
)
if write_command_line_header:
command_line = "(whatshap {}) {}".format(__version__,
" ".join(sys.argv[1:]))
else:
command_line = None
with ExitStack() as stack:
# read the given input files (BAMs, VCFs, ref...)
numeric_sample_ids = NumericSampleIds()
phased_input_reader = stack.enter_context(
PhasedInputReader(
phase_input_files,
reference,
numeric_sample_ids,
ignore_read_groups,
indels=indels,
mapq_threshold=mapping_quality,
overhang=overhang,
affine=affine_gap,
gap_start=gap_start,
gap_extend=gap_extend,
default_mismatch=mismatch,
))
show_phase_vcfs = phased_input_reader.has_vcfs
# vcf writer for final genotype likelihoods
vcf_writer = stack.enter_context(
GenotypeVcfWriter(command_line=command_line,
in_path=variant_file,
out_file=output))
# vcf writer for only the prior likelihoods (if output is desired)
prior_vcf_writer = None
if prioroutput is not None:
prior_vcf_writer = stack.enter_context(
GenotypeVcfWriter(
command_line=command_line,
in_path=variant_file,
out_file=stack.enter_context(open(prioroutput, "w")),
))
# parse vcf with input variants
# remove all likelihoods that may already be present
vcf_reader = stack.enter_context(
VcfReader(
variant_file,
indels=indels,
genotype_likelihoods=False,
ignore_genotypes=True,
))
if ignore_read_groups and not samples and len(vcf_reader.samples) > 1:
raise CommandLineError(
"When using --ignore-read-groups on a VCF with "
"multiple samples, --sample must also be used.")
if not samples:
samples = vcf_reader.samples
# if --use-ped-samples is set, use only samples from PED file
if ped and use_ped_samples:
samples = set()
for trio in PedReader(ped):
if trio.child is None or trio.mother is None or trio.father is None:
continue
samples.add(trio.mother)
samples.add(trio.father)
samples.add(trio.child)
vcf_sample_set = set(vcf_reader.samples)
for sample in samples:
if sample not in vcf_sample_set:
raise CommandLineError(
"Sample {!r} requested on command-line not found in VCF".
format(sample))
if ped and genmap:
logger.info(
"Using region-specific recombination rates from genetic map %s.",
genmap,
)
recombination_cost_computer = GeneticMapRecombinationCostComputer(
genmap)
else:
if ped:
logger.info("Using uniform recombination rate of %g cM/Mb.",
recombrate)
recombination_cost_computer = UniformRecombinationCostComputer(
recombrate)
samples = frozenset(samples)
families, family_trios = setup_families(samples, ped,
numeric_sample_ids,
max_coverage)
# Read phase information provided as VCF files, if provided.
with timers("parse_phasing_vcfs"):
phased_input_reader.read_vcfs()
# compute genotype likelihood threshold
gt_prob = 1.0 - (10**(-gt_qual_threshold / 10.0))
for variant_table in timers.iterate("parse_vcf", vcf_reader):
# create a mapping of genome positions to indices
var_to_pos = dict()
for i in range(len(variant_table.variants)):
var_to_pos[variant_table.variants[i].position] = i
chromosome = variant_table.chromosome
if (not chromosomes) or (chromosome in chromosomes):
logger.info("======== Working on chromosome %r", chromosome)
else:
logger.info(
"Leaving chromosome %r unchanged (present in VCF but not requested by option --chromosome)",
chromosome,
)
vcf_writer.write_genotypes(chromosome,
variant_table,
indels,
leave_unchanged=True)
if prioroutput is not None:
prior_vcf_writer.write_genotypes(chromosome,
variant_table,
indels,
leave_unchanged=True)
continue
positions = [v.position for v in variant_table.variants]
if not nopriors:
# compute prior genotype likelihoods based on all reads
for sample in samples:
logger.info("---- Initial genotyping of %s", sample)
with timers("read_bam"):
readset, vcf_source_ids = phased_input_reader.read(
chromosome,
variant_table.variants,
sample,
read_vcf=False,
)
readset.sort()
genotypes, genotype_likelihoods = compute_genotypes(
readset, positions)
# recompute genotypes based on given threshold
reg_genotype_likelihoods = []
for gl in range(len(genotype_likelihoods)):
norm_sum = (genotype_likelihoods[gl][0] +
genotype_likelihoods[gl][1] +
genotype_likelihoods[gl][2] +
3 * constant)
regularized = PhredGenotypeLikelihoods([
(genotype_likelihoods[gl][0] + constant) /
norm_sum,
(genotype_likelihoods[gl][1] + constant) /
norm_sum,
(genotype_likelihoods[gl][2] + constant) /
norm_sum,
])
genotypes[gl] = determine_genotype(
regularized, gt_prob)
assert isinstance(genotypes[gl], Genotype)
reg_genotype_likelihoods.append(regularized)
variant_table.set_genotype_likelihoods_of(
sample,
[
PhredGenotypeLikelihoods(list(gl))
for gl in reg_genotype_likelihoods
],
)
variant_table.set_genotypes_of(sample, genotypes)
else:
# use uniform genotype likelihoods for all individuals
for sample in samples:
variant_table.set_genotype_likelihoods_of(
sample,
[PhredGenotypeLikelihoods([1 / 3, 1 / 3, 1 / 3])] *
len(positions),
)
# if desired, output the priors in separate vcf
if prioroutput is not None:
prior_vcf_writer.write_genotypes(chromosome, variant_table,
indels)
# Iterate over all families to process, i.e. a separate DP table is created
# for each family.
for representative_sample, family in sorted(families.items()):
if len(family) == 1:
logger.info("---- Processing individual %s",
representative_sample)
else:
logger.info("---- Processing family with individuals: %s",
",".join(family))
max_coverage_per_sample = max(1, max_coverage // len(family))
logger.info("Using maximum coverage per sample of %dX",
max_coverage_per_sample)
trios = family_trios[representative_sample]
assert (len(family) == 1) or (len(trios) > 0)
# Get the reads belonging to each sample
readsets = dict()
for sample in family:
with timers("read_bam"):
readset, vcf_source_ids = phased_input_reader.read(
chromosome,
variant_table.variants,
sample,
)
with timers("select"):
readset = readset.subset([
i for i, read in enumerate(readset)
if len(read) >= 2
])
logger.info(
"Kept %d reads that cover at least two variants each",
len(readset),
)
selected_reads = select_reads(
readset,
max_coverage_per_sample,
preferred_source_ids=vcf_source_ids,
)
readsets[sample] = selected_reads
# Merge reads into one ReadSet (note that each Read object
# knows the sample it originated from).
all_reads = ReadSet()
for sample, readset in readsets.items():
for read in readset:
assert read.is_sorted(), "Add a read.sort() here"
all_reads.add(read)
all_reads.sort()
# Determine which variants can (in principle) be phased
accessible_positions = sorted(all_reads.get_positions())
logger.info(
"Variants covered by at least one phase-informative "
"read in at least one individual after read selection: %d",
len(accessible_positions),
)
# Create Pedigree
pedigree = Pedigree(numeric_sample_ids)
for sample in family:
# genotypes are assumed to be unknown, so ignore information that
# might already be present in the input vcf
all_genotype_likelihoods = variant_table.genotype_likelihoods_of(
sample)
genotype_l = [
all_genotype_likelihoods[var_to_pos[a_p]]
for a_p in accessible_positions
]
pedigree.add_individual(
sample,
[
Genotype([])
for i in range(len(accessible_positions))
],
genotype_l,
)
for trio in trios:
pedigree.add_relationship(
father_id=trio.father,
mother_id=trio.mother,
child_id=trio.child,
)
recombination_costs = recombination_cost_computer.compute(
accessible_positions)
# Finally, run genotyping algorithm
with timers("genotyping"):
problem_name = "genotyping"
logger.info(
"Genotype %d sample%s by solving the %s problem ...",
len(family),
"s" if len(family) > 1 else "",
problem_name,
)
forward_backward_table = GenotypeDPTable(
numeric_sample_ids,
all_reads,
recombination_costs,
pedigree,
accessible_positions,
)
# store results
for s in family:
likelihood_list = variant_table.genotype_likelihoods_of(
s)
genotypes_list = variant_table.genotypes_of(s)
for pos in range(len(accessible_positions)):
likelihoods = forward_backward_table.get_genotype_likelihoods(
s, pos)
# compute genotypes from likelihoods and store information
geno = determine_genotype(likelihoods, gt_prob)
assert isinstance(geno, Genotype)
genotypes_list[var_to_pos[
accessible_positions[pos]]] = geno
likelihood_list[var_to_pos[
accessible_positions[pos]]] = likelihoods
variant_table.set_genotypes_of(s, genotypes_list)
variant_table.set_genotype_likelihoods_of(
s, likelihood_list)
with timers("write_vcf"):
logger.info("======== Writing VCF")
vcf_writer.write_genotypes(chromosome, variant_table, indels)
logger.info("Done writing VCF")
logger.debug("Chromosome %r finished", chromosome)
logger.info("\n== SUMMARY ==")
total_time = timers.total()
log_memory_usage()
logger.info(
"Time spent reading BAM: %6.1f s",
timers.elapsed("read_bam"),
)
logger.info(
"Time spent parsing VCF: %6.1f s",
timers.elapsed("parse_vcf"),
)
if show_phase_vcfs:
logger.info(
"Time spent parsing input phasings from VCFs: %6.1f s",
timers.elapsed("parse_phasing_vcfs"),
)
logger.info("Time spent selecting reads: %6.1f s",
timers.elapsed("select"))
logger.info(
"Time spent genotyping: %6.1f s",
timers.elapsed("genotyping"),
)
logger.info(
"Time spent writing VCF: %6.1f s",
timers.elapsed("write_vcf"),
)
logger.info(
"Time spent on rest: %6.1f s",
total_time - timers.sum(),
)
logger.info("Total elapsed time: %6.1f s",
total_time)
def run_polyphase(
phase_input_files,
variant_file,
ploidy,
reference=None,
output=sys.stdout,
samples=None,
chromosomes=None,
verify_genotypes=False,
ignore_read_groups=False,
indels=True,
mapping_quality=20,
tag="PS",
include_haploid_sets=False,
write_command_line_header=True,
read_list_filename=None,
ce_bundle_edges=False,
min_overlap=2,
plot_clusters=False,
plot_threading=False,
ce_refinements=5,
block_cut_sensitivity=4,
):
"""
Run Polyploid Phasing.
phase_input_files -- list of paths to BAM/CRAM/VCF files
variant-file -- path to input VCF
reference -- path to reference FASTA
output -- path to output VCF or a file like object
samples -- names of samples to phase. An empty list means: phase all samples
chromosomes -- names of chromosomes to phase. An empty list means: phase all chromosomes
ignore_read_groups
mapping_quality -- discard reads below this mapping quality
tag -- How to store phasing info in the VCF, can be 'PS' or 'HP'
write_command_line_header -- whether to add a ##commandline header to the output VCF
"""
timers = StageTimer()
logger.info(
"This is WhatsHap (polyploid) %s running under Python %s",
__version__,
platform.python_version(),
)
numeric_sample_ids = NumericSampleIds()
with ExitStack() as stack:
assert phase_input_files
phased_input_reader = stack.enter_context(
PhasedInputReader(
phase_input_files,
reference,
numeric_sample_ids,
ignore_read_groups,
indels=indels,
mapq_threshold=mapping_quality,
))
assert not phased_input_reader.has_vcfs
if write_command_line_header:
command_line = "(whatshap {}) {}".format(__version__,
" ".join(sys.argv[1:]))
else:
command_line = None
try:
vcf_writer = stack.enter_context(
PhasedVcfWriter(
command_line=command_line,
in_path=variant_file,
out_file=output,
tag=tag,
ploidy=ploidy,
include_haploid_sets=include_haploid_sets,
))
except OSError as e:
raise CommandLineError(e)
vcf_reader = stack.enter_context(
VcfReader(
variant_file,
indels=indels,
phases=True,
genotype_likelihoods=False,
ploidy=ploidy,
))
if ignore_read_groups and not samples and len(vcf_reader.samples) > 1:
raise CommandLineError(
"When using --ignore-read-groups on a VCF with "
"multiple samples, --sample must also be used.")
if not samples:
samples = vcf_reader.samples
vcf_sample_set = set(vcf_reader.samples)
for sample in samples:
if sample not in vcf_sample_set:
raise CommandLineError(
"Sample {!r} requested on command-line not found in VCF".
format(sample))
if block_cut_sensitivity < 0:
logger.warning(
"Block cut sensitivity was set to negative value. Lowest value (0) is assumed instead."
)
block_cut_sensitivity = 0
elif block_cut_sensitivity > 5:
logger.warning(
"Block cut sensitivity level too large. Assuming highest valid value (5) instead."
)
block_cut_sensitivity = 5
samples = frozenset(samples)
read_list_file = None
if read_list_filename:
raise NotImplementedError("create_read_list_file not implemented")
# read_list_file = create_read_list_file(read_list_filename)
# Store phasing parameters in tuple to keep function signatures cleaner
phasing_param = PhasingParameter(
ploidy=ploidy,
verify_genotypes=verify_genotypes,
ce_bundle_edges=ce_bundle_edges,
min_overlap=min_overlap,
ce_refinements=ce_refinements,
block_cut_sensitivity=block_cut_sensitivity,
plot_clusters=plot_clusters,
plot_threading=plot_threading,
)
timers.start("parse_vcf")
try:
for variant_table in vcf_reader:
chromosome = variant_table.chromosome
timers.stop("parse_vcf")
if (not chromosomes) or (chromosome in chromosomes):
logger.info("======== Working on chromosome %r",
chromosome)
else:
logger.info(
"Leaving chromosome %r unchanged (present in VCF but not requested by option --chromosome)",
chromosome,
)
with timers("write_vcf"):
superreads, components = dict(), dict()
vcf_writer.write(chromosome, superreads, components)
continue
# These two variables hold the phasing results for all samples
superreads, components, haploid_components = dict(), dict(
), dict()
# Iterate over all samples to process
for sample in samples:
logger.info("---- Processing individual %s", sample)
# Process inputs for this sample
missing_genotypes = set()
heterozygous = set()
genotypes = variant_table.genotypes_of(sample)
for index, gt in enumerate(genotypes):
if gt.is_none():
missing_genotypes.add(index)
elif not gt.is_homozygous():
heterozygous.add(index)
else:
assert gt.is_homozygous()
to_discard = set(range(
len(variant_table))).difference(heterozygous)
phasable_variant_table = deepcopy(variant_table)
# Remove calls to be discarded from variant table
phasable_variant_table.remove_rows_by_index(to_discard)
logger.info(
"Number of variants skipped due to missing genotypes: %d",
len(missing_genotypes),
)
logger.info(
"Number of remaining heterozygous variants: %d",
len(phasable_variant_table))
# Get the reads belonging to this sample
timers.start("read_bam")
readset, vcf_source_ids = phased_input_reader.read(
chromosome, phasable_variant_table.variants, sample)
readset.sort()
timers.stop("read_bam")
# Verify genotypes
if verify_genotypes:
timers.start("verify_genotypes")
logger.info("Verify genotyping of %s", sample)
positions = [
v.position for v in phasable_variant_table.variants
]
computed_genotypes = [
Genotype(gt) for gt in compute_polyploid_genotypes(
readset, ploidy, positions)
]
# skip all positions at which genotypes do not match
given_genotypes = phasable_variant_table.genotypes_of(
sample)
matching_genotypes = []
missing_genotypes = set()
print(computed_genotypes, len(computed_genotypes))
print(given_genotypes, len(given_genotypes))
print(len(positions))
for i, g in enumerate(given_genotypes):
c_g = computed_genotypes[i]
if (g == c_g) or (c_g is None):
matching_genotypes.append(g)
else:
matching_genotypes.append(Genotype([]))
missing_genotypes.add(i)
phasable_variant_table.set_genotypes_of(
sample, matching_genotypes)
# Remove variants with deleted genotype
phasable_variant_table.remove_rows_by_index(
missing_genotypes)
logger.info(
"Number of variants removed due to inconsistent genotypes: %d",
len(missing_genotypes),
)
logger.info(
"Number of remaining heterozygous variants: %d",
len(phasable_variant_table),
)
# Re-read the readset to remove discarded variants
readset, vcf_source_ids = phased_input_reader.read(
chromosome, phasable_variant_table.variants,
sample)
readset.sort()
timers.stop("verify_genotypes")
# Remove reads with insufficient variants
readset = readset.subset([
i for i, read in enumerate(readset)
if len(read) >= max(2, min_overlap)
])
logger.info(
"Kept %d reads that cover at least two variants each",
len(readset))
# Adapt the variant table to the subset of reads
phasable_variant_table.subset_rows_by_position(
readset.get_positions())
# Run the actual phasing
(
sample_components,
sample_haploid_components,
sample_superreads,
) = phase_single_individual(readset,
phasable_variant_table, sample,
phasing_param, output, timers)
# Collect results
components[sample] = sample_components
haploid_components[sample] = sample_haploid_components
superreads[sample] = sample_superreads
with timers("write_vcf"):
logger.info("======== Writing VCF")
vcf_writer.write(
chromosome,
superreads,
components,
haploid_components if include_haploid_sets else None,
)
# TODO: Use genotype information to polish results
# assert len(changed_genotypes) == 0
logger.info("Done writing VCF")
logger.debug("Chromosome %r finished", chromosome)
timers.start("parse_vcf")
timers.stop("parse_vcf")
except PloidyError as e:
raise CommandLineError(e)
if read_list_file:
read_list_file.close()
logger.info("\n== SUMMARY ==")
log_memory_usage()
logger.info("Time spent reading BAM/CRAM: %6.1f s",
timers.elapsed("read_bam"))
logger.info("Time spent parsing VCF: %6.1f s",
timers.elapsed("parse_vcf"))
if verify_genotypes:
logger.info(
"Time spent verifying genotypes: %6.1f s",
timers.elapsed("verify_genotypes"),
)
logger.info("Time spent detecting blocks: %6.1f s",
timers.elapsed("detecting_blocks"))
logger.info("Time spent scoring reads: %6.1f s",
timers.elapsed("read_scoring"))
logger.info(
"Time spent solving cluster editing: %6.1f s",
timers.elapsed("solve_clusterediting"),
)
logger.info("Time spent threading haplotypes: %6.1f s",
timers.elapsed("threading"))
if plot_clusters or plot_threading:
logger.info("Time spent creating plots: %6.1f s",
timers.elapsed("create_plots"))
logger.info("Time spent writing VCF: %6.1f s",
timers.elapsed("write_vcf"))
logger.info("Time spent on rest: %6.1f s",
timers.total() - timers.sum())
logger.info("Total elapsed time: %6.1f s",
timers.total())
def run_whatshap(
phase_input_files,
variant_file,
reference=None,
output=sys.stdout,
samples=None,
chromosomes=None,
ignore_read_groups=False,
indels=True,
mapping_quality=20,
read_merging=False,
read_merging_error_rate=0.15,
read_merging_max_error_rate=0.25,
read_merging_positive_threshold=1000000,
read_merging_negative_threshold=1000,
max_coverage=15,
full_genotyping=False,
distrust_genotypes=False,
include_homozygous=False,
ped=None,
recombrate=1.26,
genmap=None,
genetic_haplotyping=True,
recombination_list_filename=None,
tag="PS",
read_list_filename=None,
gl_regularizer=None,
gtchange_list_filename=None,
default_gq=30,
write_command_line_header=True,
use_ped_samples=False,
algorithm="whatshap",
):
"""
Run WhatsHap.
phase_input_files -- list of paths to BAM/CRAM/VCF files
variant_file -- path to input VCF
reference -- path to reference FASTA
output -- path to output VCF or a file-like object
samples -- names of samples to phase. an empty list means: phase all samples
chromosomes -- names of chromosomes to phase. an empty list means: phase all chromosomes
ignore_read_groups
mapping_quality -- discard reads below this mapping quality
read_merging -- whether or not to merge reads
read_merging_error_rate -- probability that a nucleotide is wrong
read_merging_max_error_rate -- max error rate on edge of merge graph considered
read_merging_positive_threshold -- threshold on the ratio of the two probabilities
read_merging_negative_threshold -- threshold on the opposite ratio of positive threshold
max_coverage
full_genotyping
distrust_genotypes
include_homozygous
genetic_haplotyping -- in ped mode, merge disconnected blocks based on genotype status
recombination_list_filename -- filename to write putative recombination events to
tag -- How to store phasing info in the VCF, can be 'PS' or 'HP'
read_list_filename -- name of file to write list of used reads to
algorithm -- algorithm to use, can be 'whatshap' or 'hapchat'
gl_regularizer -- float to be passed as regularization constant to GenotypeLikelihoods.as_phred
gtchange_list_filename -- filename to write list of changed genotypes to
default_gq -- genotype likelihood to be used when GL or PL not available
write_command_line_header -- whether to add a ##commandline header to the output VCF
"""
if algorithm == "hapchat" and ped is not None:
raise CommandLineError(
"The hapchat algorithm cannot do pedigree phasing")
timers = StageTimer()
logger.info(
"This is WhatsHap %s running under Python %s",
__version__,
platform.python_version(),
)
if full_genotyping:
distrust_genotypes = True
include_homozygous = True
numeric_sample_ids = NumericSampleIds()
if write_command_line_header:
command_line = "(whatshap {}) {}".format(__version__,
" ".join(sys.argv[1:]))
else:
command_line = None
if read_merging:
read_merger = ReadMerger(
read_merging_error_rate,
read_merging_max_error_rate,
read_merging_positive_threshold,
read_merging_negative_threshold,
)
else:
read_merger = DoNothingReadMerger()
with ExitStack() as stack:
try:
vcf_writer = stack.enter_context(
PhasedVcfWriter(
command_line=command_line,
in_path=variant_file,
out_file=output,
tag=tag,
))
except (OSError, VcfError) as e:
raise CommandLineError(e)
phased_input_reader = stack.enter_context(
PhasedInputReader(
phase_input_files,
reference,
numeric_sample_ids,
ignore_read_groups,
mapq_threshold=mapping_quality,
indels=indels,
))
show_phase_vcfs = phased_input_reader.has_vcfs
# Only read genotype likelihoods from VCFs when distrusting genotypes
vcf_reader = stack.enter_context(
VcfReader(variant_file,
indels=indels,
genotype_likelihoods=distrust_genotypes))
if ignore_read_groups and not samples and len(vcf_reader.samples) > 1:
raise CommandLineError(
"When using --ignore-read-groups on a VCF with "
"multiple samples, --sample must also be used.")
if not samples:
samples = vcf_reader.samples
# if --use-ped-samples is set, use only samples from PED file
if ped and use_ped_samples:
samples = PedReader(ped).samples()
raise_if_any_sample_not_in_vcf(vcf_reader, samples)
if ped and genmap:
logger.info(
"Using region-specific recombination rates from genetic map %s.",
genmap,
)
try:
recombination_cost_computer = GeneticMapRecombinationCostComputer(
genmap)
except ParseError as e:
raise CommandLineError(e)
else:
if ped:
logger.info("Using uniform recombination rate of %g cM/Mb.",
recombrate)
recombination_cost_computer = UniformRecombinationCostComputer(
recombrate)
samples = frozenset(samples)
families, family_trios = setup_families(samples, ped,
numeric_sample_ids,
max_coverage)
read_list = None
if read_list_filename:
read_list = stack.enter_context(ReadList(read_list_filename))
if algorithm == "hapchat":
logger.warning(
"On which haplotype a read occurs in the inferred solution is not yet "
"implemented in hapchat, and so the corresponding column in the "
"read list file contains no information about this")
with timers("parse_phasing_vcfs"):
# TODO should this be done in PhasedInputReader.__init__?
phased_input_reader.read_vcfs()
for variant_table in timers.iterate("parse_vcf", vcf_reader):
chromosome = variant_table.chromosome
if (not chromosomes) or (chromosome in chromosomes):
logger.info("======== Working on chromosome %r", chromosome)
else:
logger.info(
"Leaving chromosome %r unchanged (present in VCF but not requested by option --chromosome)",
chromosome,
)
with timers("write_vcf"):
superreads, components = dict(), dict()
vcf_writer.write(chromosome, superreads, components)
continue
if full_genotyping:
positions = [v.position for v in variant_table.variants]
for sample in samples:
logger.info("---- Initial genotyping of %s", sample)
with timers("read_bam"):
bam_sample = None if ignore_read_groups else sample
readset, vcf_source_ids = phased_input_reader.read(
chromosome,
variant_table.variants,
bam_sample,
read_vcf=False,
)
readset.sort() # TODO can be removed
genotypes, genotype_likelihoods = compute_genotypes(
readset, positions)
variant_table.set_genotypes_of(sample, genotypes)
variant_table.set_genotype_likelihoods_of(
sample,
[
GenotypeLikelihoods(gl)
for gl in genotype_likelihoods
],
)
# These two variables hold the phasing results for all samples
superreads, components = dict(), dict()
# Iterate over all families to process, i.e. a separate DP table is created
# for each family.
# TODO: Can the body of this loop be factored out into a phase_family function?
for representative_sample, family in sorted(families.items()):
if len(family) == 1:
logger.info("---- Processing individual %s",
representative_sample)
else:
logger.info("---- Processing family with individuals: %s",
",".join(family))
max_coverage_per_sample = max(1, max_coverage // len(family))
logger.info("Using maximum coverage per sample of %dX",
max_coverage_per_sample)
trios = family_trios[representative_sample]
assert len(family) == 1 or len(trios) > 0
homozygous_positions, phasable_variant_table = find_phaseable_variants(
family, include_homozygous, trios, variant_table)
# Get the reads belonging to each sample
readsets = dict() # TODO this could become a list
for sample in family:
with timers("read_bam"):
readset, vcf_source_ids = phased_input_reader.read(
chromosome,
phasable_variant_table.variants,
sample,
)
# TODO: Read selection done w.r.t. all variants, where using heterozygous
# variants only would probably give better results.
with timers("select"):
readset = readset.subset([
i for i, read in enumerate(readset)
if len(read) >= 2
])
logger.info(
"Kept %d reads that cover at least two variants each",
len(readset),
)
merged_reads = read_merger.merge(readset)
selected_reads = select_reads(
merged_reads,
max_coverage_per_sample,
preferred_source_ids=vcf_source_ids,
)
readsets[sample] = selected_reads
if len(family) == 1 and not distrust_genotypes:
# When having a pedigree (len(family) > 1), blocks are also merged after
# phasing based on the pedigree information and these statistics are not
# so useful. When distrust_genotypes, genotypes can change during phasing
# and so can the block structure. So don't print these stats in those cases
log_best_case_phasing_info(readset, selected_reads)
all_reads = merge_readsets(readsets)
# Determine which variants can (in principle) be phased
accessible_positions = sorted(all_reads.get_positions())
logger.info(
"Variants covered by at least one phase-informative "
"read in at least one individual after read selection: %d",
len(accessible_positions),
)
if len(family) > 1 and genetic_haplotyping:
# In case of genetic haplotyping, also retain all positions homozygous
# in at least one individual (because they might be phased based on genotypes)
accessible_positions = sorted(
set(accessible_positions).union(homozygous_positions))
logger.info(
"Variants either covered by phase-informative read or homozygous "
"in at least one individual: %d",
len(accessible_positions),
)
# Keep only accessible positions
phasable_variant_table.subset_rows_by_position(
accessible_positions)
assert len(phasable_variant_table.variants) == len(
accessible_positions)
pedigree = create_pedigree(
default_gq,
distrust_genotypes,
family,
gl_regularizer,
numeric_sample_ids,
phasable_variant_table,
trios,
)
recombination_costs = recombination_cost_computer.compute(
accessible_positions)
# Finally, run phasing algorithm
with timers("phase"):
problem_name = "MEC" if len(family) == 1 else "PedMEC"
logger.info(
"Phasing %d sample%s by solving the %s problem ...",
len(family),
plural_s(len(family)),
problem_name,
)
if algorithm == "hapchat":
dp_table = HapChatCore(all_reads)
else:
dp_table = PedigreeDPTable(
all_reads,
recombination_costs,
pedigree,
distrust_genotypes,
accessible_positions,
)
superreads_list, transmission_vector = dp_table.get_super_reads(
)
optimal_cost = dp_table.get_optimal_cost()
logger.info("%s cost: %d", problem_name, optimal_cost)
with timers("components"):
master_block = None
heterozygous_positions_by_sample = None
# If we distrusted genotypes, we need to re-determine which sites are h**o-/heterozygous after phasing
if distrust_genotypes:
hom_in_any_sample = set()
heterozygous_positions_by_sample = {}
heterozygous_gts = frozenset({(0, 1), (1, 0)})
homozygous_gts = frozenset({(0, 0), (1, 1)})
for sample, sample_superreads in zip(
family, superreads_list):
hets = set()
for v1, v2 in zip(*sample_superreads):
assert v1.position == v2.position
if v1.position not in accessible_positions:
continue
gt = (v1.allele, v2.allele)
if gt in heterozygous_gts:
hets.add(v1.position)
elif gt in homozygous_gts:
hom_in_any_sample.add(v1.position)
heterozygous_positions_by_sample[
numeric_sample_ids[sample]] = hets
if len(family) > 1 and genetic_haplotyping:
master_block = sorted(hom_in_any_sample)
else:
if len(family) > 1 and genetic_haplotyping:
master_block = sorted(
set(homozygous_positions).intersection(
set(accessible_positions)))
overall_components = find_components(
accessible_positions,
all_reads,
master_block,
heterozygous_positions_by_sample,
)
n_phased_blocks = len(set(overall_components.values()))
logger.info("No. of phased blocks: %d", n_phased_blocks)
largest_component = find_largest_component(
overall_components)
if len(largest_component) > 0:
logger.info(
"Largest component contains %d variants (%.1f%% of accessible variants) between position %d and %d",
len(largest_component),
len(largest_component) * 100.0 /
len(accessible_positions),
largest_component[0] + 1,
largest_component[-1] + 1,
)
if recombination_list_filename:
n_recombinations = write_recombination_list(
recombination_list_filename,
chromosome,
accessible_positions,
overall_components,
recombination_costs,
transmission_vector,
trios,
)
logger.info(
"Total no. of detected recombination events: %d",
n_recombinations,
)
# Superreads in superreads_list are in the same order as individuals were added to the pedigree
for sample, sample_superreads in zip(family, superreads_list):
superreads[sample] = sample_superreads
assert len(sample_superreads) == 2
assert (sample_superreads[0].sample_id ==
sample_superreads[1].sample_id ==
numeric_sample_ids[sample])
# identical for all samples
components[sample] = overall_components
if read_list:
read_list.write(
all_reads,
dp_table.get_optimal_partitioning(),
components,
numeric_sample_ids,
)
with timers("write_vcf"):
logger.info("======== Writing VCF")
changed_genotypes = vcf_writer.write(chromosome, superreads,
components)
logger.info("Done writing VCF")
if changed_genotypes:
assert distrust_genotypes
logger.info("Changed %d genotypes while writing VCF",
len(changed_genotypes))
if gtchange_list_filename:
logger.info("Writing list of changed genotypes to %r",
gtchange_list_filename)
write_changed_genotypes(gtchange_list_filename,
changed_genotypes)
logger.debug("Chromosome %r finished", chromosome)
log_time_and_memory_usage(timers, show_phase_vcfs=show_phase_vcfs)