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calculate_coverage_v3.py
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calculate_coverage_v3.py
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import argparse
import pysam
import sys
from collections import defaultdict
from concurrent.futures import ProcessPoolExecutor
def get_fragment_from_read(
read, extend_reads, center_reads, default_fragment_length,
max_paired_fragment_length
):
"""
Get read start and end position of a read in the style of deepTools
`get_fragment_from_read`.
"""
if not extend_reads and default_fragment_length == 'read length':
return read.get_blocks()
if not extend_reads:
return [(read.reference_start, read.reference_end)]
if (
read.is_paired and read.is_proper_pair
) and (
abs(read.template_length) < max_paired_fragment_length
):
if read.is_reverse:
fragment_start = read.next_reference_start
fragment_end = read.reference_end
else:
fragment_start = read.reference_start
fragment_end = read.reference_start + abs(read.template_length)
else:
if read.is_reverse:
fragment_start = read.reference_end - default_fragment_length
fragment_end = read.reference_end
else:
fragment_start = read.reference_start
fragment_end = read.reference_start + default_fragment_length
if center_reads:
fragment_center = (
fragment_end - (fragment_end - fragment_start) / 2
)
fragment_start = int(
fragment_center - read.infer_query_length(always=False) / 2
)
fragment_end = (
fragment_start + read.infer_query_length(always=False)
)
assert fragment_start < fragment_end, f"Fragment start greater than fragment end for read {read.query_name}"
return [(fragment_start, fragment_end)]
def parse_bam_file(
bam_file_path, sam_flag_include, min_mapq, extend_reads, center_reads,
default_fragment_length, max_paired_fragment_length
):
bam_entries = defaultdict(list)
with pysam.AlignmentFile(bam_file_path, 'rb') as bam_file:
for read in bam_file.fetch():
if (
read.flag & sam_flag_include
) and (
read.mapping_quality >= min_mapq
):
chrom = bam_file.get_reference_name(read.reference_id)
fragments = get_fragment_from_read(
read,
extend_reads,
center_reads,
default_fragment_length,
max_paired_fragment_length
)
for fragment_start, fragment_end in fragments:
frag_length = fragment_end - fragment_start
bam_entries[chrom].append(
(fragment_start, fragment_end, frag_length)
)
return bam_entries
def calculate_coverage_for_chromosome(
chromosome, bam_entries, total_reads, bin_size, normalize
):
if bin_size <= 0:
raise ValueError("bin_size must be greater than 0")
coverage = defaultdict(float)
for start, end, frag_length in bam_entries:
contribution = 1 / frag_length if normalize else 1
for pos in range(start, end + 1):
coverage[(chromosome, pos)] += contribution
if normalize:
for key in coverage:
coverage[key] /= total_reads
binned_coverage = defaultdict(float)
for (chrom, pos), value in coverage.items():
bin_start = (pos // bin_size) * bin_size
binned_coverage[(chrom, bin_start)] += value / bin_size
return binned_coverage
def calculate_coverage_task(data):
return calculate_coverage_for_chromosome(*data)
def write_bedgraph(coverage, output_file, bin_size):
with open(output_file, 'w') as outfile:
for (chrom, bin_start), value in sorted(
coverage.items(), key=lambda x: (x[0][0], x[0][1])
):
output_line = (
f"{chrom}\t{bin_start}\t{bin_start + bin_size}\t{value:.24f}\n"
)
outfile.write(output_line)
def main():
parser = argparse.ArgumentParser(
description=(
'Calculate binned coverage, normalized or not, from BAM file.'
),
formatter_class=argparse.ArgumentDefaultsHelpFormatter
)
parser.add_argument(
'-i', '--input_file',
required=True,
help='Path to the input BAM file.'
)
parser.add_argument(
'-o', '--output_file',
required=True,
help='Path to the output BEDGRAPH file.'
)
parser.add_argument(
'-b', '--bin_size',
type=int,
default=30,
help='Bin size for coverage calculation in base pairs.'
)
parser.add_argument(
'-t', '--threads',
type=int,
default=1,
help='Number of threads for parallel processing.'
)
parser.add_argument(
'-n', '--normalize',
action='store_true',
help=(
'Normalize coverage by fragment length and total reads, ' +
'generating "normalized coverage" as described in Dickson et ' +
'al., Sci Rep 2023. If not specified, then "typical/raw ' +
'coverage" is calculated.'
)
)
parser.add_argument(
'-s', '--SAM_flag_include',
type=int,
default=0,
help='SAM flag to include reads.'
)
parser.add_argument(
'-m', '--min_MAPQ',
type=int,
default=0,
help='Minimum mapping quality (MAPQ).'
)
parser.add_argument(
'-e', '--extend_reads',
action='store_true',
help='Extend reads to fragment length (in the manner of deepTools).'
)
parser.add_argument(
'-c', '--center_reads',
action='store_true',
help=(
'Center reads on fragment length (in the manner of ' +
'deepTools).'
)
)
parser.add_argument(
'-d', '--default_fragment_length',
type=int,
default=200,
help='Default fragment length for extending reads.'
)
parser.add_argument(
'-l', '--max_paired_fragment_length',
type=int,
default=1000,
help='Maximum fragment length for considering "proper pairs."'
)
if len(sys.argv) == 1:
parser.print_help(sys.stderr)
sys.exit(1)
args = parser.parse_args()
print("# Parameters --------------------------")
print(f" Input file: {args.input_file}")
print(f" Output file: {args.output_file}")
print(f" Bin size: {args.bin_size}")
print(f" Threads: {args.threads}")
print(f" Normalize: {args.normalize}")
print(f" SAM flag include: {args.SAM_flag_include}")
print(f" Minimum MAPQ: {args.min_MAPQ}")
print(f" Extend reads: {args.extend_reads}")
print(f" Center reads: {args.center_reads}")
print(f" Default fragment length: {args.default_fragment_length}")
print(f"Max paired fragment length: {args.max_paired_fragment_length}")
if args.bin_size <= 0:
raise ValueError("bin_size must be greater than 0")
bam_entries = parse_bam_file(
args.input_file,
args.SAM_flag_include,
args.min_MAPQ,
args.extend_reads,
args.center_reads,
args.default_fragment_length,
args.max_paired_fragment_length
)
total_reads = sum(len(entries) for entries in bam_entries.values())
combined_coverage = defaultdict(float)
task_data = [
(chrom, entries, total_reads, args.bin_size, args.normalize)
for chrom, entries in bam_entries.items()
]
with ProcessPoolExecutor(max_workers=args.threads) as executor:
results = executor.map(calculate_coverage_task, task_data)
for result in results:
for key, value in result.items():
combined_coverage[key] += value
write_bedgraph(combined_coverage, args.output_file, args.bin_size)
if __name__ == "__main__":
main()