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extract_UTR_lengths_stop_to_polyA.py
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extract_UTR_lengths_stop_to_polyA.py
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#!/usr/bin/env python3
import pysam
import re
import os
import logging
from Bio import SeqIO
import pandas as pd
import argparse
def get_args():
"""
Parse command-line arguments.
"""
parser = argparse.ArgumentParser(description="Extract poly(A) sites and calculate distance from stop codon.",
add_help=False)
optional = parser.add_argument_group('optional arguments')
optional.add_argument("--bam", dest='bam',
action="store",
nargs='+',
required=True,
type=str,
help="List of BAM files to be parsed.")
optional.add_argument("--output", dest='output',
action="store",
type=str,
required=True,
help="Directory to store output files.")
optional.add_argument("--fasta", dest='fasta',
action="store",
type=str,
required=True,
help="FASTA file of the reference genome.")
optional.add_argument("--reference_transcript", dest='reference_transcript',
action="store",
type=str,
required=True,
help="FASTA file of the reference transcripts with UTR.")
optional.add_argument("--polyA_length", dest='polyA_length',
action="store",
type=int,
default=7,
help="Minimum length of poly(A) tail to consider.")
optional.add_argument("--log", dest='log',
action="store",
type=str,
default="extract_polA_site.log",
help="Log file to store the logging information.")
parser.add_argument("-h", "--help",
action="help",
default=argparse.SUPPRESS,
help="Show this help message and exit.")
return parser.parse_args()
def index_reference_transcripts(reference_transcript_file):
"""
Index reference transcripts from a FASTA file.
Parameters:
reference_transcript_file (str): Path to the FASTA file containing reference transcript sequences.
Returns:
dict: A dictionary of reference transcripts indexed by transcript ID.
"""
return SeqIO.to_dict(SeqIO.parse(reference_transcript_file, "fasta"))
def find_stop_codon_position(ref_seq, reference_transcript):
"""
Find the stop codon position in the reference sequence.
Parameters:
ref_seq (Seq): Reference sequence as a Biopython Seq object.
reference_transcript (SeqRecord): Reference transcript as a Biopython SeqRecord object.
Returns:
int: Position of the stop codon in the reference sequence.
Raises:
ValueError: If the reference transcript is not found in the reference sequence.
"""
ref_seq_str = str(ref_seq)
reference_transcript_str = str(reference_transcript.seq)
match_start = ref_seq_str.find(reference_transcript_str)
if match_start == -1:
raise ValueError(f"Reference transcript not found in sequence for {reference_transcript.id}")
stop_codon_position = match_start + len(reference_transcript_str)
return stop_codon_position
def find_polyA_from_right(seq, polyA_length):
"""
Search for poly(A) sequences from the right-hand side of the read.
This function searches for a poly(A) tail starting from the right-hand (3') side of the read.
If a poly(A) sequence is found, it returns the start position of the poly(A) tail in the read.
Parameters:
seq (str): The read sequence.
polyA_length (int): Minimum length of poly(A) tail to consider.
Returns:
int: The start position of the poly(A) tail in the read sequence.
"""
# Search for poly(A) stretch at the end of the sequence
right_match = re.search(r'(A{' + str(polyA_length) + ',})$', seq)
if right_match:
# Return the start position of the poly(A) within the read
return right_match.start()
else:
return None
def extract_polyA_sites_with_distance(bam_file, fasta_file, reference_transcripts,
polyA_length, output_dir):
"""
Extract poly(A) sites from a BAM file and calculate distance from stop codon.
Parameters:
bam_file (str): Path to the BAM file to be processed.
fasta_file (str): Path to the FASTA file containing reference genome sequences.
reference_transcripts (dict): Dictionary of reference transcripts indexed by transcript ID.
polyA_length (int): Minimum length of poly(A) tail to consider.
output_dir (str): Directory to output the results.
Returns:
None
"""
logging.info(f"Processing file: {bam_file}")
bam = pysam.AlignmentFile(bam_file, "rb")
fasta = SeqIO.to_dict(SeqIO.parse(fasta_file, "fasta"))
output_file = os.path.join(output_dir, f"{os.path.basename(bam_file)}_polyA_distances.tsv")
with open(output_file, 'w') as out_f:
out_f.write("transcript_ID\tread_name\tdistance_to_polyA\n")
for read in bam.fetch():
if not read.is_unmapped:
seq = read.query_sequence
transcript_id = read.reference_name
if transcript_id not in reference_transcripts:
continue
ref_seq = fasta[transcript_id].seq
reference_transcript = reference_transcripts[transcript_id]
try:
stop_codon_position = find_stop_codon_position(ref_seq, reference_transcript)
except ValueError as e:
logging.debug(e)
continue
# Only consider poly(A) sites after the stop codon
if read.reference_start < stop_codon_position:
match = re.search(r'(A{' + str(polyA_length) + ',})', seq)
if match:
start_pos = match.start()
coordinate = read.reference_start + start_pos
# Search again after stop codon if match within CDS
if coordinate < stop_codon_position:
match = re.search(r'(A{' + str(polyA_length) + ',})',
seq[stop_codon_position - read.reference_start:])
if match:
start_pos = match.start() + (stop_codon_position - read.reference_start)
coordinate = read.reference_start + start_pos
if match and coordinate >= stop_codon_position:
read_name = read.query_name
distance_to_stop = coordinate - stop_codon_position
out_f.write(f"{transcript_id}\t{read_name}\t{distance_to_stop}\n")
if match and coordinate >= stop_codon_position:
read_name = read.query_name
polyA_start = read.reference_start + start_pos
polyA_length_detected = len(match.group(0))
distance_to_stop = coordinate - stop_codon_position
old_polyA_start = polyA_start
# Step 2: Call the new function to check for poly(A) from the right side
right_start_pos = find_polyA_from_right(seq, polyA_length)
# If a valid right-side polyA site is found and positions differ, update the start position
if right_start_pos is not None and right_start_pos != start_pos:
logging.info(f"Right-side poly(A) site found at different position for read {read_name}. Updating poly(A) start position.")
polyA_start = read.reference_start + right_start_pos
coordinate = read.reference_start + right_start_pos # Update the coordinate to the new polyA start
distance_to_stop = coordinate - stop_codon_position # Recalculate distance to stop
# Update the sequence preceding the polyA tail from the read based on the new right-hand polyA start
pre_polyA_seq_from_read = seq[max(0, right_start_pos - distance_to_stop):right_start_pos]
# Update the sequence preceding the polyA tail from reference genome
pre_polyA_seq_from_ref = str(fasta[transcript_id].seq[stop_codon_position:coordinate]) if distance_to_stop > 0 else ""
out_f.write(f"{transcript_id}\t{read_name}\t{distance_to_stop}\n")
# Sequence from reference genome
pre_polyA_seq_from_ref = str(fasta[transcript_id].seq[stop_codon_position:coordinate]) if distance_to_stop > 0 else ""
bam.close()
logging.info(f"Processed {bam_file} and saved results to {output_file}")
def main():
"""
Main function to handle the overall script execution.
"""
args = get_args()
# Setup logging
logging.basicConfig(level=logging.INFO,
format='%(asctime)s - %(levelname)s - %(message)s',
filename=args.log, filemode='w')
console = logging.StreamHandler()
console.setLevel(logging.INFO)
logging.getLogger('').addHandler(console)
# Index the reference transcripts
reference_transcripts = index_reference_transcripts(args.reference_transcript)
output_dir = args.output # Directory to store outputs
# Process each BAM file
for bam_file in args.bam:
extract_polyA_sites_with_distance(bam_file, args.fasta, reference_transcripts,
args.polyA_length, output_dir)
if __name__ == "__main__":
main()