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Copy pathSVIndelGenotyper.py
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SVIndelGenotyper.py
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#!/usr/bin/env python3
import os
import sys
import re
import subprocess
import json
import math
import random
import string
# Define constant paths and folders
blast_exe_folder="./tool/"
def print_usage():
print(f"Usage: python {sys.argv[0]} [SV_list_fn] [optional: clean_deepseq_folder]")
sys.exit()
def get_bit_fn(bam, reference_folder="./reference/"):
bit_fn = reference_folder + "hg19.fa"
genome_version = "hg19"
chr_in_bam = "yes"
genome_version_command = f"samtools idxstats {bam} | head -1"
chr_checkresult = str(subprocess.check_output(genome_version_command, shell=True), "utf-8")
if "chr1\t248956422" in chr_checkresult or "1\t248956422" in chr_checkresult:
bit_fn = reference_folder + "hg38.fa"
genome_version = "hg38"
if chr_checkresult.count("chr") == 0:
chr_in_bam = "no"
return bit_fn, genome_version, chr_in_bam
def get_revcmp(strs):
cpm = {"A": "T", "T": "A", "C": "G", "G": "C", "N": "N", "": "", "-": "-"}
rev = strs[::-1]
revcmp = "".join([cpm[i] for i in rev])
return revcmp
def get_depth_indel(indel, sample, chr_in_bam, bam, clean_deepseq_folder="no"):
chro, pos, ref_allele, mut_allele = indel.split(".")
if clean_deepseq_folder != "no":
clean_deepseq_fn = f"{clean_deepseq_folder}/{sample}.count.gz"
depth_pos_command = f"tabix {clean_deepseq_fn} {chro.replace('chr','')}:{pos}-{pos}"
depth_pos = "0"
try:
pos_result = str(subprocess.check_output(depth_pos_command, shell=True), "utf-8").strip()
if len(pos_result) > 0:
depth_pos = pos_result.split("\n")[0].split("\t")[-1]
except:
print(f"no depth: {chro}.{pos}")
else:
if chr_in_bam == "yes":
depth_pos_command = f"samtools depth -r {chro}:{pos}-{pos} {bam} | cut -f3"
else:
depth_pos_command = f"samtools depth -r {chro.replace('chr', '')}:{pos}-{pos} {bam} | cut -f3"
try:
depth_pos = str(subprocess.check_output(depth_pos_command, shell=True), "utf-8").strip()
except:
print(f"no depth: {chro}.{pos}")
return depth_pos
def make_ref_indel(indel, bam, bit_fn):
chro, pos, ref_allele, mut_allele = indel.split(".")
sample = os.path.basename(bam)
if "chr" not in indel:
chro = "chr" + chro
startA = int(pos) - 200
endA = int(pos) - 1
startB = int(pos) + len(ref_allele)
endB = int(startB) + 200 - 1
left_ref_command = f"samtools faidx {bit_fn} {chro}:{startA}-{endA}"
right_ref_command = f"samtools faidx {bit_fn} {chro}:{startB}-{endB}"
left_ref_seq = ("").join(str(subprocess.check_output(left_ref_command, shell=True), "utf-8").strip().split("\n")[1:]).upper()
right_ref_seq = ("").join(str(subprocess.check_output(right_ref_command, shell=True), "utf-8").strip().split("\n")[1:]).upper()
WT_ref_seq = left_ref_seq + ref_allele + right_ref_seq
mut_ref_seq = left_ref_seq + mut_allele + right_ref_seq
WT_genome_pos = [f"{chro}.{i}" for i in range(startA, endB + 1)]
mut_genome_pos = [f"{chro}.{i}" for i in range(startA, endA + 2)] + [f"mut.{i}" for i in range(len(mut_allele) - 1)] + [f"{chro}.{i}" for i in range(startB, endB + 1)]
WT_ref_fn = ''.join(random.choice(string.ascii_lowercase) for _ in range(7)) + "." + sample + "." + indel + "_WT_ref.fa"
mut_ref_fn = ''.join(random.choice(string.ascii_lowercase) for _ in range(7)) + "." + sample + "." + indel + "_mut_ref.fa"
with open(WT_ref_fn, "w") as WT_ref_fh:
WT_ref_fh.write(">" + sample + "." + indel + "\n")
WT_ref_fh.write(WT_ref_seq + "\n")
with open(mut_ref_fn, "w") as mut_ref_fh:
mut_ref_fh.write(">" + sample + "." + indel + "\n")
mut_ref_fh.write(mut_ref_seq + "\n")
ref_data = {
"WT_ref_fn": WT_ref_fn,
"mut_ref_fn": mut_ref_fn,
"WT_ref_seq": WT_ref_seq,
"mut_ref_seq": mut_ref_seq,
"WT_genome_pos": WT_genome_pos,
"mut_genome_pos": mut_genome_pos,
"WT_keyseq": WT_ref_seq[(200 - 6):(200 + 6)],
"WT_keyseq_pos": WT_genome_pos[(200 - 6):(200 + 6)],
"mut_keyseq": mut_ref_seq[(200 - 6):(200 + len(mut_allele) + 6)],
"mut_keyseq_pos": mut_genome_pos[(200 - 6):(200 + len(mut_allele) + 6)]
}
return ref_data
def extract_sam_indel(indel, bam, chr_in_bam):
chro, pos, ref_allele, mut_allele = indel.split(".")
sample = os.path.basename(bam).replace(".bam", "")
get_chro_command = f"samtools idxstats {bam} | cut -f1"
chr_checkresult = str(subprocess.check_output(get_chro_command, shell=True), "utf-8")
if chr_in_bam == "yes" and "chr" not in indel:
chro = "chr" + chro
if chr_in_bam == "no" and "chr" in indel:
chro = chro.replace("chr", "")
sam_fn = ''.join(random.choice(string.ascii_lowercase) for _ in range(8)) + "." + indel + "." + os.path.basename(bam).replace(".bam", ".sam")
command_pos = f"samtools view {bam} {chro}:{int(pos) - 100}-{int(pos) + 100} | sort -k1,1 > {sam_fn}"
os.system(command_pos)
return sam_fn
def get_read_qual(qual_str, qual_cutoff):
read_len = len(qual_str)
ngood = 0
for nn_qual in qual_str:
qscore = ord(nn_qual) - 33
if qscore > qual_cutoff:
ngood += 1
qual_perc = round(float(ngood) / read_len, 3)
return qual_perc
def extract_fasta(sam_fn, qual_perc_cutoff):
seq_data = {}
seqid_data = {}
candidate_fa_fn = sam_fn.replace(".sam", ".candidate.fa")
candidate_fa_fh = open(candidate_fa_fn, 'w')
with open(sam_fn, 'r') as sam_fh:
for line in sam_fh:
lst = line.strip().split('\t')
seqid = lst[0] + "_" + lst[1]
chro = lst[2]
pos = lst[3]
map_qual = lst[4]
cigar = lst[5]
paired_read_chr = lst[6]
paired_read_pos = lst[7]
segment_length = lst[8]
seq = lst[9].upper()
qual_str = lst[10]
ngood = 0
if re.findall("^\d+M$", cigar):
continue
Q20_qual_perc = get_read_qual(qual_str, 20)
Q30_qual_perc = get_read_qual(qual_str, 30)
if Q20_qual_perc < qual_perc_cutoff:
continue
if seq not in seq_data:
seq_data[seq] = {}
candidate_fa_fh.write(">" + seqid + "\n")
candidate_fa_fh.write(seq + "\n")
seqid_data[seqid] = seq
seq_data[seq][seqid] = {"20": Q20_qual_perc, "30": Q30_qual_perc}
candidate_fa_fh.close()
return seq_data, seqid_data, candidate_fa_fn
def do_blast_indel(WT_ref_fn, mut_ref_fn, candidate_fa_fn):
WT_blast_fn = WT_ref_fn.replace(".fa", ".blast")
mut_blast_fn = mut_ref_fn.replace(".fa", ".blast")
command_WT = f"{blast_exe_folder}blastn -task blastn -word_size 4 -evalue 0.0001 -penalty -3 -reward 2 -gapopen 6 -gapextend 2 -dust no -soft_masking false -query {candidate_fa_fn} -subject {WT_ref_fn} -outfmt 15 > {WT_blast_fn}"
command_mut = f"{blast_exe_folder}blastn -task blastn -word_size 4 -evalue 0.0001 -penalty -3 -reward 2 -gapopen 6 -gapextend 2 -dust no -soft_masking false -query {candidate_fa_fn} -subject {mut_ref_fn} -outfmt 15 > {mut_blast_fn}"
os.system(command_WT)
os.system(command_mut)
return WT_blast_fn, mut_blast_fn
def adjust_indel_pos(qseq, hseq, midline):
hseq_lst = list(hseq)
qseq_lst = list(qseq)
midline_lst = list(midline)
for i in range(len(qseq_lst)):
if qseq_lst[i] == hseq_lst[i]:
continue
if hseq_lst[i] == "-" and i < len(hseq_lst) - 1:
j = 1
while hseq_lst[i + j] == "-" and (i + j) < len(hseq_lst) - 1:
j += 1
if (i + j) <= (len(hseq_lst) - 1) and hseq_lst[i + j] == qseq_lst[i]:
hseq_lst[i] = hseq_lst[i + j]
hseq_lst[i + j] = "-"
midline_lst[i] = "|"
midline_lst[i + j] = " "
else:
break
if qseq_lst[i] == "-" and (i < len(qseq_lst) - 1):
j = 1
while (qseq_lst[i + j] == "-") and (i + j) < len(qseq_lst) - 1:
j += 1
if (i + j) <= (len(qseq_lst) - 1) and qseq_lst[i + j] == hseq_lst[i]:
qseq_lst[i] = qseq_lst[i + j]
qseq_lst[i + j] = "-"
midline_lst[i] = "|"
midline_lst[i + j] = " "
else:
break
adjust_qseq = "".join(qseq_lst)
adjust_hseq = "".join(hseq_lst)
adjust_midline = "".join(midline_lst)
return adjust_qseq, adjust_hseq, adjust_midline
def adjust_1seg_indel_pos(seqid, qseq, hseq, midline, geno_pos_inalign, indel, blast_trim = 8):
hseq_lst = list(hseq)
qseq_lst = list(qseq)
midline_lst = list(midline)
for i in range(len(qseq_lst)):
if qseq_lst[i] == hseq_lst[i]:
continue
if hseq_lst[i] == "-" and i < len(hseq_lst) - 1:
j = 1
while hseq_lst[i + j] == "-" and (i + j) < len(hseq_lst) - 1:
j += 1
if (i + j) <= (len(hseq_lst) - 1) and hseq_lst[i + j] == qseq_lst[i]:
hseq_lst[i] = hseq_lst[i + j]
hseq_lst[i + j] = "-"
midline_lst[i] = "|"
midline_lst[i + j] = " "
else:
break
if qseq_lst[i] == "-" and (i < len(qseq_lst) - 1):
j = 1
while (qseq_lst[i + j] == "-") and (i + j) < len(qseq_lst) - 1:
j += 1
if (i + j) <= (len(qseq_lst) - 1) and qseq_lst[i + j] == hseq_lst[i]:
qseq_lst[i] = qseq_lst[i + j]
qseq_lst[i + j] = "-"
midline_lst[i] = "|"
midline_lst[i + j] = " "
else:
break
qseq = "".join(qseq_lst)
hseq = "".join(hseq_lst)
midline = "".join(midline_lst)
left_index = 0
for ii in range(len(qseq_lst)):
if qseq_lst[ii] == hseq_lst[ii]:
continue
if not (hseq_lst[ii] == "-" or qseq_lst[ii] == "-"):
continue
left_index = ii - 1
break
left_qseq = "".join(qseq_lst[0:(left_index + 1)])
left_hseq = "".join(hseq_lst[0:(left_index + 1)])
left_midline = "".join(midline_lst[0:(left_index + 1)])
while (left_index - blast_trim + 1 >= 0) and (left_midline[(left_index - blast_trim + 1):(left_index + 1)] != "|" * blast_trim):
left_index = left_index - 1
indel_left_genome_pos_in_align = []
left_breakpoint_geno_pos = ".".join(indel.split(".")[0:2])
j = 0
for bb in hseq:
if bb == "-":
indel_left_genome_pos_in_align.append("-")
continue
indel_left_genome_pos_in_align.append(geno_pos_inalign[j])
j += 1
try:
left_breakpoint_index_inalign = indel_left_genome_pos_in_align.index(left_breakpoint_geno_pos)
if left_breakpoint_index_inalign < left_index:
left_index = left_breakpoint_index_inalign
left_qseq = "".join(qseq_lst[0:(left_index + 1)])
left_hseq = "".join(hseq_lst[0:(left_index + 1)])
left_midline = "".join(midline_lst[0:(left_index + 1)])
right_index = left_index + 1
right_qseq_rev, right_hseq_rev, right_midline_rev = adjust_indel_pos(qseq[right_index:][::-1], hseq[right_index:][::-1], midline[right_index:][::-1])
right_qseq, right_hseq, right_midline = right_qseq_rev[::-1], right_hseq_rev[::-1], right_midline_rev[::-1]
adjust_qseq = left_qseq + right_qseq
adjust_hseq = left_hseq + right_hseq
adjust_midline = left_midline + right_midline
keyseq_left_index = left_breakpoint_index_inalign - 6
keyseq_right_index = left_breakpoint_index_inalign + len(indel.split(".")[-1]) + 6
key_qseq = adjust_qseq[keyseq_left_index:keyseq_right_index]
key_hseq = adjust_hseq[keyseq_left_index:keyseq_right_index]
except:
adjust_qseq, adjust_hseq, adjust_midline, key_qseq, key_hseq = ["ND", "ND", "ND", "ND", "ND"]
return adjust_qseq, adjust_hseq, adjust_midline, key_qseq, key_hseq
def parse_blast_sv(blast_fn, SV):
strand_transform={"Plus":"+","Minus":"-","ND":"ND"}
blast_json=json.load(open(blast_fn))["BlastOutput2"]
blast_data={}
for i in range(0,len(blast_json)):
blast_report= blast_json[i]["report"]
blast_result= blast_report["results"]
bl2seq=blast_result["bl2seq"][0]
seqid=bl2seq["query_title"]
if seqid not in blast_data:
blast_data[seqid]={}
query_len=bl2seq["query_len"]
hsps="ND"
align_len="0"
identity="0"
gaps="ND"
query_from="ND"
query_to="ND"
query_strand="ND"
hit_from="ND"
hit_to="ND"
hit_strand="ND"
qseq="ND"
hseq="ND"
midline="ND"
bit_score="ND"
score="0"
evalue="ND"
if ("hits" in bl2seq) and len(bl2seq["hits"])>0:
hits=bl2seq["hits"]
hsps=hits[0]["hsps"][0]
align_len=hsps["align_len"]
identity=hsps["identity"]
gaps=hsps["gaps"]
query_from=hsps["query_from"]
query_to=hsps["query_to"]
query_strand=hsps["query_strand"]
hit_from=hsps["hit_from"]
hit_to=hsps["hit_to"]
hit_strand=hsps["hit_strand"]
qseq=hsps["qseq"]
hseq=hsps["hseq"]
midline=hsps["midline"]
bit_score=hsps["bit_score"]
score=hsps["score"]
evalue=hsps["evalue"]
blast_data[seqid]={
"initial_SV":SV,
"align_len":align_len,
"identity":identity,
"gaps":gaps,
"query_from":query_from,
"query_to":query_to,
"query_strand":strand_transform[query_strand],
"hit_from":hit_from,
"hit_to":hit_to,
"hit_strand":strand_transform[hit_strand],
"qseq":qseq,
"hseq":hseq,
"midline":midline,
"bit_score":bit_score,
"score":score,
"evalue":evalue
}
return blast_data
def parse_blast_indel(blast_fn, indel):
strand_transform = {"Plus": "+", "Minus": "-", "ND": "ND"}
blast_json = json.load(open(blast_fn))["BlastOutput2"]
blast_data = {}
for i in range(0, len(blast_json)):
blast_report = blast_json[i]["report"]
blast_result = blast_report["results"]
bl2seq = blast_result["bl2seq"][0]
seqid = bl2seq["query_title"]
if seqid not in blast_data:
blast_data[seqid] = {}
query_len = bl2seq["query_len"]
hsps = bl2seq.get("hits", [{"hsps": [{}]}])[0]["hsps"][0]
align_len = hsps.get("align_len", "0")
identity = hsps.get("identity", "0")
gaps = hsps.get("gaps", "ND")
query_from = hsps.get("query_from", "ND")
query_to = hsps.get("query_to", "ND")
query_strand = strand_transform.get(hsps.get("query_strand"), "ND")
hit_from = hsps.get("hit_from", "ND")
hit_to = hsps.get("hit_to", "ND")
hit_strand = strand_transform.get(hsps.get("hit_strand"), "ND")
qseq = hsps.get("qseq", "ND")
hseq = hsps.get("hseq", "ND")
midline = hsps.get("midline", "ND")
bit_score = hsps.get("bit_score", "ND")
score = hsps.get("score", "0")
evalue = hsps.get("evalue", "ND")
blast_data[seqid] = {
"initial_indel": indel,
"align_len": align_len,
"identity": identity,
"gaps": gaps,
"query_from": query_from,
"query_to": query_to,
"query_strand": query_strand,
"hit_from": hit_from,
"hit_to": hit_to,
"hit_strand": hit_strand,
"qseq": qseq,
"hseq": hseq,
"midline": midline,
"bit_score": bit_score,
"score": score,
"evalue": evalue
}
return blast_data
def genotyping_indel(WT_blast_data, mut_blast_data, seqid_data, seq_data, ref_data, indel, output_qual_pairs):
WT_ref_fn = ref_data["WT_ref_fn"]
mut_ref_fn = ref_data["mut_ref_fn"]
WT_ref_seq = ref_data["WT_ref_seq"]
mut_ref_seq = ref_data["mut_ref_seq"]
WT_genome_pos = ref_data["WT_genome_pos"]
mut_genome_pos = ref_data["mut_genome_pos"]
WT_keyseq = ref_data["WT_keyseq"]
mut_keyseq = ref_data["mut_keyseq"]
WT_keyseq_pos = ref_data["WT_keyseq_pos"]
mut_keyseq_pos = ref_data["mut_keyseq_pos"]
revcmp_WT_ref_seq = get_revcmp(WT_ref_seq)
revcmp_mut_ref_seq = get_revcmp(mut_ref_seq)
revcmp_WT_keyseq = get_revcmp(WT_keyseq)
revcmp_mut_keyseq = get_revcmp(mut_keyseq)
indel_read_ids = {"exact": [], "fuzzy": []}
indel_read_num = {}
for output_qual_pair in output_qual_pairs:
indel_read_num["exact_" + output_qual_pair] = {}
indel_read_num["fuzzy_" + output_qual_pair] = {}
for seqid in seqid_data:
is_indel = "no"
seq = seqid_data[seqid]
all_seqids = seq_data[seq]
WT_blast_info = WT_blast_data[seqid]
mut_blast_info = mut_blast_data[seqid]
WT_score = WT_blast_info["score"]
mut_score = mut_blast_info["score"]
WT_align_len = WT_blast_info["align_len"]
mut_align_len = mut_blast_info["align_len"]
WT_gaps = WT_blast_info["gaps"]
mut_gaps = mut_blast_info["gaps"]
WT_qseq = WT_blast_info["qseq"]
mut_qseq = mut_blast_info["qseq"]
WT_midline = WT_blast_info["midline"]
mut_midline = mut_blast_info["midline"]
WT_hseq = WT_blast_info["hseq"]
mut_hseq = mut_blast_info["hseq"]
WT_identity = mut_blast_info["identity"]
mut_identity = mut_blast_info["identity"]
WT_hit_strand = WT_blast_info["hit_strand"]
mut_hit_strand = mut_blast_info["hit_strand"]
mut_query_from = mut_blast_info["query_from"]
mut_query_to = mut_blast_info["query_to"]
mut_hit_from = mut_blast_info["hit_from"]
mut_hit_to = mut_blast_info["hit_to"]
WT_query_from = WT_blast_info["query_from"]
WT_query_to = WT_blast_info["query_to"]
seqid_score_lst = [(int(WT_score), int(WT_identity), "WT"), (int(mut_score), int(mut_identity), "indel")]
sorted_seqid_score_lst = sorted(seqid_score_lst, key=lambda x: x[0], reverse=True)
if sorted_seqid_score_lst[0][2] != "indel":
continue
if sorted_seqid_score_lst[0][1] < len(seq) * 0.95:
continue
if mut_hit_strand == "+":
mut_genomepos_inalign = mut_genome_pos[(mut_hit_from - 1):mut_hit_to]
if not (mut_keyseq in mut_qseq.replace("-", "") and mut_keyseq in mut_hseq.replace("-", "")):
continue
mut_qseq, mut_hseq, mut_midline, key_qseq_in_align, key_hseq_in_align = adjust_1seg_indel_pos(seqid, mut_qseq, mut_hseq, mut_midline, mut_genomepos_inalign, indel)
if "ND" in mut_qseq:
continue
if mut_keyseq == key_hseq_in_align == key_qseq_in_align:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[0:-1])
indel_read_num["exact_" + output_qual_pair][unique_seqidi] = 1
indel_read_ids["exact"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
else:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[0:-1])
indel_read_num["fuzzy_" + output_qual_pair][unique_seqidi] = 1
indel_read_ids["fuzzy"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
if mut_hit_strand == "-":
updated_mut_qseq = get_revcmp(mut_qseq)
updated_mut_midline = mut_midline[::-1]
updated_mut_hseq = get_revcmp(mut_hseq)
updated_mut_query_from = mut_query_to
updated_mut_query_to = mut_query_from
updated_mut_hit_from = mut_hit_to
updated_mut_hit_to = mut_hit_from
mut_genomepos_inalign = mut_genome_pos[(updated_mut_hit_from - 1):updated_mut_hit_to]
if not (mut_keyseq in updated_mut_qseq.replace("-", "") and mut_keyseq in updated_mut_hseq.replace("-", "")):
continue
mut_qseq, updated_mut_hseq, updated_mut_midline, key_qseq_in_align, key_hseq_in_align = adjust_1seg_indel_pos(seqid, updated_mut_qseq, updated_mut_hseq, updated_mut_midline, mut_genomepos_inalign, indel)
if "ND" in mut_qseq:
continue
if mut_keyseq == key_hseq_in_align == key_qseq_in_align:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[0:-1])
indel_read_num["exact_" + output_qual_pair][unique_seqidi] = 1
indel_read_ids["exact"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
else:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[0:-1])
indel_read_num["fuzzy_" + output_qual_pair][unique_seqidi] = 1
indel_read_ids["fuzzy"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
exact_indel_read_ids = indel_read_ids["exact"]
fuzzy_indel_read_ids = indel_read_ids["fuzzy"]
exact_indel_read_num = [len(indel_read_num["exact_" + op]) for op in output_qual_pairs]
fuzzy_indel_read_num = [(len(indel_read_num["fuzzy_" + op]) + len(indel_read_num["exact_" + op])) for op in output_qual_pairs]
return (exact_indel_read_ids, fuzzy_indel_read_ids, exact_indel_read_num, fuzzy_indel_read_num)
def process_indel(indel_line, output_qual_pairs, centromere_regions, clean_deepseq_folder, qual_perc_cutoff, out_indel_reads_fh):
lst = indel_line.strip().split("\t")
initial_indel = lst[0]
indel = lst[1]
indel_padding = lst[2]
bam = lst[3]
sample = os.path.basename(bam).replace(".bam", "")
bit_fn, genome_version, chr_in_bam = get_bit_fn(bam)
chro, pos, ref_allele, mut_allele = indel.split(".")
out_indel = (f"{sample}\t{initial_indel}\t{indel}\t{indel_padding}\tND"+ "\tND" * len(output_qual_pairs)+ "\tND\n")
if chro not in centromere_regions[genome_version] or (
centromere_regions[genome_version][chro][0] < int(pos) < centromere_regions[genome_version][chro][1]
):
return out_indel
sam_fn = extract_sam_indel(indel, bam, chr_in_bam)
depth_pos = get_depth_indel(indel, sample, chr_in_bam, bam, clean_deepseq_folder)
seq_data, seqid_data, candidate_fa_fn = extract_fasta(sam_fn, qual_perc_cutoff)
candidate_fa_fn_check_command = f"wc -l {candidate_fa_fn}"
candidate_fa_fn_check_result = str(
subprocess.check_output(candidate_fa_fn_check_command, shell=True), "utf-8"
)
files_to_delete = [sam_fn, candidate_fa_fn]
if candidate_fa_fn_check_result.split(" ")[0] == "0":
return (f"{out_indel}{sample}\t{initial_indel}\t{indel}\t{indel_padding}\t0"+ "\t0" * len(output_qual_pairs)+ f"\t{depth_pos}\n")
print(" #reads: ", candidate_fa_fn_check_result.split(" ")[0])
ref_data = make_ref_indel(indel, bam, bit_fn)
WT_ref_fn = ref_data["WT_ref_fn"]
mut_ref_fn = ref_data["mut_ref_fn"]
WT_blast_fn, mut_blast_fn = do_blast_indel(WT_ref_fn, mut_ref_fn, candidate_fa_fn)
files_to_delete.extend([WT_ref_fn, mut_ref_fn, WT_blast_fn, mut_blast_fn])
WT_blast_data = parse_blast_indel(WT_blast_fn, indel)
mut_blast_data = parse_blast_indel(mut_blast_fn, indel)
(
exact_indel_read_ids,
fuzzy_indel_read_ids,
exact_indel_read_num,
fuzzy_indel_read_num,
) = genotyping_indel(
WT_blast_data, mut_blast_data, seqid_data, seq_data, ref_data, indel, output_qual_pairs
)
exact_indel_read_num_str = '\t'.join(map(str, exact_indel_read_num))
fuzzy_indel_read_num_str = '\t'.join(map(str, fuzzy_indel_read_num))
out_indel = (
sample + "\t" + initial_indel + "\t" + indel + "\t" + indel_padding + "\t" +
exact_indel_read_num_str + "\t" + fuzzy_indel_read_num_str + "\t" + depth_pos + "\n"
)
out_indel_reads = ""
for read_id in exact_indel_read_ids:
out_indel_reads_fh.write(initial_indel+"\t"+indel+"\t"+indel_padding+"\t"+("\t").join(read_id)+"\t"+bam+"\texact_match\n")
for read_id in fuzzy_indel_read_ids:
out_indel_reads_fh.write(initial_indel+"\t"+indel+"\t"+indel_padding+"\t"+("\t").join(read_id)+"\t"+bam+"\tfuzzy_match\n")
# Remove files
for file_to_delete in set(files_to_delete):
if os.path.isfile(file_to_delete):
os.remove(file_to_delete)
return f"{out_indel}{out_indel_reads}"
def adjust_SV(SV):
if SV.count(".") == 5:
SV += "."
cmp_strand = {"+": "-", "-": "+"}
SV_lst = SV.split(".")
chrA = SV_lst[0]
if "chr" not in chrA:
chrA = "chr" + chrA
if chrA == "chrX":
chrA = "chr23"
if chrA == "chrY":
chrA = "chr24"
posA = SV_lst[1]
strandA = SV_lst[2]
chrB = SV_lst[3]
if "chr" not in chrB:
chrB = "chr" + chrB
if chrB == "chrX":
chrB = "chr23"
if chrB == "chrY":
chrB = "chr24"
posB = SV_lst[4]
strandB = SV_lst[5]
insertion = SV_lst[6]
adjusted_chrA = chrA
adjusted_posA = posA
adjusted_strandA = strandA
adjusted_chrB = chrB
adjusted_posB = posB
adjusted_strandB = strandB
adjusted_insertion = insertion
if (
int(chrA.replace("chr", "")) > int(chrB.replace("chr", ""))
or (
int(chrA.replace("chr", ""))
== int(chrB.replace("chr", ""))
and int(posA) > int(posB)
)
):
adjusted_chrA = chrB
adjusted_posA = posB
adjusted_strandA = cmp_strand[strandB]
adjusted_chrB = chrA
adjusted_posB = posA
adjusted_strandB = cmp_strand[strandA]
adjusted_insertion = get_revcmp(insertion)
if adjusted_chrA == "chr23":
adjusted_chrA = "chrX"
if adjusted_chrA == "chr24":
adjusted_chrA = "chrY"
if adjusted_chrB == "chr23":
adjusted_chrB = "chrX"
if adjusted_chrB == "chr24":
adjusted_chrB = "chrY"
adjusted_SV = f"{adjusted_chrA}.{adjusted_posA}.{adjusted_strandA}.{adjusted_chrB}.{adjusted_posB}.{adjusted_strandB}.{adjusted_insertion}"
return adjusted_SV
def get_depth_sv(SV, sample, chr_in_bam, bam, clean_deepseq_folder="no"):
chrA, posA, strandA, chrB, posB, strandB, insertion = SV.split(".")
depth_posA = "0"
depth_posB = "0"
def get_depth_command(chr, pos, bam):
return (
f"samtools depth -r {chr.replace('chr', '')}:{pos}-{pos} {bam}|cut -f3"
)
def get_tabix_command(file_path, chr, pos):
return f"tabix {file_path} {chr.replace('chr', '')}:{pos}-{pos}"
if not clean_deepseq_folder == "no":
clean_deepseq_fn = f"{clean_deepseq_folder}/{sample}.count.gz"
try:
posA_result = (
str(subprocess.check_output(get_tabix_command(clean_deepseq_fn, chrA, posA), shell=True), "utf-8").strip()
)
if len(posA_result) > 0:
depth_posA = posA_result.split("\n")[0].split("\t")[-1]
except:
print(f"no depth: {chrA}.{posA}")
try:
posB_result = (
str(subprocess.check_output(get_tabix_command(clean_deepseq_fn, chrB, posB), shell=True), "utf-8").strip()
)
if len(posB_result) > 0:
depth_posB = posB_result.split("\n")[0].split("\t")[-1]
except:
print(f"no depth: {chrB}.{posB}")
else:
depth_posA_command = get_depth_command(chrA, posA, bam)
depth_posB_command = get_depth_command(chrB, posB, bam)
try:
depth_posA = str(subprocess.check_output(depth_posA_command, shell=True), "utf-8").strip()
except:
print(f"no depth: {chrA}.{posA}")
try:
depth_posB = str(subprocess.check_output(depth_posB_command, shell=True), "utf-8").strip()
except:
print(f"no depth: {chrB}.{posB}")
if depth_posA == "":
depth_posA = "0"
if depth_posB == "":
depth_posB = "0"
return depth_posA, depth_posB
def make_ref_sv(SV, bam, bit_fn):
SV_lst = SV.split(".")
chrA, posA, strandA, chrB, posB, strandB, insertion = SV_lst
sample = os.path.basename(bam)
if "chr" not in SV:
chrA = "chr" + chrA
chrB = "chr" + chrB
startA = int(posA) - 200
endA = int(posA) + 200
startB = int(posB) - 200
endB = int(posB) + 200
posA_ref_command = f"samtools faidx {bit_fn} {chrA}:{startA}-{endA}"
posB_ref_command = f"samtools faidx {bit_fn} {chrB}:{startB}-{endB}"
posA_ref_seq = ("" .join(str(subprocess.check_output(posA_ref_command, shell=True), "utf-8").strip().split("\n")[1:])).upper()
posB_ref_seq = ("" .join(str(subprocess.check_output(posB_ref_command, shell=True), "utf-8").strip().split("\n")[1:])).upper()
posA_genome_pos = [f"{chrA}.{i}" for i in range(startA, endA + 1)]
posB_genome_pos = [f"{chrB}.{i}" for i in range(startB, endB + 1)]
insertion_pos = [f"ins.{i}" for i in range(len(insertion))]
if strandA == "+" and strandB == "-":
posB_ref_seq = get_revcmp(posB_ref_seq)
posB_genome_pos = posB_genome_pos[::-1]
if strandA == "-" and strandB == "+":
posA_ref_seq = get_revcmp(posA_ref_seq)
posA_genome_pos = posA_genome_pos[::-1]
if strandA == "-" and strandB == "-":
posA_ref_seq = get_revcmp(posA_ref_seq)
posA_genome_pos = posA_genome_pos[::-1]
posB_ref_seq = get_revcmp(posB_ref_seq)
posB_genome_pos = posB_genome_pos[::-1]
SV_ref_seq = posA_ref_seq[0:201] + insertion + posB_ref_seq[200:]
SV_genome_pos = posA_genome_pos[0:201] + insertion_pos + posB_genome_pos[200:]
posA_ref_fn = "".join(random.choice(string.ascii_lowercase) for x in range(7)) + "." + sample + "." + chrA + "." + posA + "." + strandA + "_ref.fa"
posB_ref_fn = "".join(random.choice(string.ascii_lowercase) for x in range(6)) + "." + sample + "." + chrB + "." + posB + "." + strandB + "_ref.fa"
SV_ref_fn = "".join(random.choice(string.ascii_lowercase) for x in range(8)) + "." + sample + "." + SV + "_ref.fa"
with open(posA_ref_fn, "w") as posA_ref_fh:
posA_ref_fh.write(f">{sample}.{chrA}.{posA}.{strandA}\n")
posA_ref_fh.write(posA_ref_seq + "\n")
with open(posB_ref_fn, "w") as posB_ref_fh:
posB_ref_fh.write(f">{sample}.{chrB}.{posB}.{strandB}\n")
posB_ref_fh.write(posB_ref_seq + "\n")
with open(SV_ref_fn, "w") as SV_ref_fh:
SV_ref_fh.write(f">{sample}.{SV}\n")
SV_ref_fh.write(SV_ref_seq + "\n")
ref_data = {
"posA_ref_fn": posA_ref_fn,
"posB_ref_fn": posB_ref_fn,
"SV_ref_fn": SV_ref_fn,
"posA_ref_seq": posA_ref_seq,
"posB_ref_seq": posB_ref_seq,
"SV_ref_seq": SV_ref_seq,
"insertion_seq": insertion,
"posA_genome_pos": posA_genome_pos,
"posB_genome_pos": posB_genome_pos,
"SV_genome_pos": SV_genome_pos,
"insertion_pos": insertion_pos,
"posA_keyseq": posA_ref_seq[(200 - 6):(200 + 6)],
"posA_keyseq_pos": posA_genome_pos[(200 - 6):(200 + 6)],
"posB_keyseq": posB_ref_seq[(200 - 6):(200 + 6)],
"posB_keyseq_pos": posB_genome_pos[(200 - 6):(200 + 6)],
"SV_keyseq": SV_ref_seq[(200 - 6):(200 + len(insertion) + 6)],
"SV_keyseq_pos": SV_genome_pos[(200 - 6):(200 + len(insertion) + 6)]
}
return ref_data
def extract_sam_sv(SV, bam, chr_in_bam):
SV_lst = SV.split(".")
chrA, posA, strandA, chrB, posB, strandB, _ = SV_lst
sample = os.path.basename(bam).replace(".bam", "")
def get_chromosome(chr_in_bam, chr):
if chr_in_bam == "yes" and ("chr" not in chr):
return "chr" + chr
elif chr_in_bam == "no" and ("chr" in chr):
return chr.replace("chr", "")
return chr
chrA = get_chromosome(chr_in_bam, chrA)
chrB = get_chromosome(chr_in_bam, chrB)
sam_fn = ('').join(random.choice(string.ascii_lowercase) for x in range(8)) + f".{SV}.{sample}.sam"
command_A = f"samtools view {bam} {chrA}:{int(posA) - 100}-{int(posA) + 100} | sort -k1,1 > {sam_fn}"
command_B = f"samtools view {bam} {chrB}:{int(posB) - 100}-{int(posB) + 100} | sort -k1,1 >> {sam_fn}"
os.system(command_A)
os.system(command_B)
return sam_fn
def do_blast_sv(posA_ref_fn, posB_ref_fn, SV_ref_fn, candidate_fa_fn):
posA_blast_fn = posA_ref_fn.replace(".fa", ".blast")
posB_blast_fn = posB_ref_fn.replace(".fa", ".blast")
SV_blast_fn = SV_ref_fn.replace(".fa", ".blast")
blast_command = (
"{}blastn -task blastn -word_size 4 -evalue 0.0001 -penalty -3 -reward 2 "
"-gapopen 6 -gapextend 2 -dust no -soft_masking false -query {} "
"-subject {} -outfmt 15 > {}"
)
os.system(blast_command.format(blast_exe_folder, candidate_fa_fn, posA_ref_fn, posA_blast_fn))
os.system(blast_command.format(blast_exe_folder, candidate_fa_fn, posB_ref_fn, posB_blast_fn))
os.system(blast_command.format(blast_exe_folder, candidate_fa_fn, SV_ref_fn, SV_blast_fn))
return (posA_blast_fn, posB_blast_fn, SV_blast_fn)
def genotyping_SV(posA_blast_data, posB_blast_data, SV_blast_data, seqid_data, seq_data, ref_data, SV, output_qual_pairs):
posA_ref_fn, posB_ref_fn, SV_ref_fn = ref_data["posA_ref_fn"], ref_data["posB_ref_fn"], ref_data["SV_ref_fn"]
posA_ref_seq, posB_ref_seq, SV_ref_seq = ref_data["posA_ref_seq"], ref_data["posB_ref_seq"], ref_data["SV_ref_seq"]
insertion_seq, posA_genome_pos, posB_genome_pos = ref_data["insertion_seq"], ref_data["posA_genome_pos"], ref_data["posB_genome_pos"]
SV_genome_pos, insertion_pos = ref_data["SV_genome_pos"], ref_data["posA_genome_pos"]
posA_keyseq, posB_keyseq, SV_keyseq = ref_data["posA_keyseq"], ref_data["posB_keyseq"], ref_data["SV_keyseq"]
posA_keyseq_pos, posB_keyseq_pos, SV_keyseq_pos = ref_data["posA_keyseq_pos"], ref_data["posB_keyseq_pos"], ref_data["SV_keyseq_pos"]
SV_read_ids = {"exact": [], "fuzzy": []}
SV_read_num = {}
for output_qual_pair in output_qual_pairs:
SV_read_num[f"exact_{output_qual_pair}"] = {}
SV_read_num[f"fuzzy_{output_qual_pair}"] = {}
for seqid in seqid_data:
seq = seqid_data[seqid]
all_seqids = seq_data[seq]
posA_blast_info, posB_blast_info, SV_blast_info = posA_blast_data[seqid], posB_blast_data[seqid], SV_blast_data[seqid]
posA_score, posB_score, SV_score = posA_blast_info["score"], posB_blast_info["score"], SV_blast_info["score"]
posA_identity, posB_identity, SV_identity = posA_blast_info["identity"], posB_blast_info["identity"], SV_blast_info["identity"]
posA_qseq, posB_qseq, SV_qseq = posA_blast_info["qseq"], posB_blast_info["qseq"], SV_blast_info["qseq"]
posA_midline, posB_midline, SV_midline = posA_blast_info["midline"], posB_blast_info["midline"], SV_blast_info["midline"]
posA_hseq, posB_hseq, SV_hseq = posA_blast_info["hseq"], posB_blast_info["hseq"], SV_blast_info["hseq"]
posA_hit_strand, posB_hit_strand, SV_hit_strand = posA_blast_info["hit_strand"], posB_blast_info["hit_strand"], SV_blast_info["hit_strand"]
SV_query_from, SV_query_to, SV_hit_from, SV_hit_to = SV_blast_info["query_from"], SV_blast_info["query_to"], SV_blast_info["hit_from"], SV_blast_info["hit_to"]
seqid_score_lst = [(int(posA_score), int(posA_identity), "posA"), (int(posB_score), int(posB_identity), "posB"), (int(SV_score), int(SV_identity), "SV")]
sorted_seqid_score_lst = sorted(seqid_score_lst, key=lambda x: x[0], reverse=True)
if sorted_seqid_score_lst[0][2] != "SV":
continue
if sorted_seqid_score_lst[0][1] < len(seq) * 0.95:
continue
SV_genomepos_inalign = SV_genome_pos[(SV_hit_from - 1):SV_hit_to]
if SV_hit_strand == "+":
if SV_keyseq not in SV_qseq.replace("-", "") or SV_keyseq not in SV_hseq.replace("-", ""):
continue
SV_qseq, SV_hseq, SV_midline, key_qseq_in_align, key_hseq_in_align = adjust_1seg_indel_pos(seqid, SV_qseq, SV_hseq, SV_midline, SV_genomepos_inalign, SV)
if "ND" in SV_qseq:
continue
if SV_keyseq == key_hseq_in_align == key_qseq_in_align:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[:-1])
SV_read_num[f"exact_{output_qual_pair}"][unique_seqidi] = 1
SV_read_ids["exact"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
else:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[:-1])
SV_read_num[f"fuzzy_{output_qual_pair}"][unique_seqidi] = 1
SV_read_ids["fuzzy"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
elif SV_hit_strand == "-":
updated_SV_qseq = get_revcmp(SV_qseq)
updated_SV_midline = SV_midline[::-1]
updated_SV_hseq = get_revcmp(SV_hseq)
updated_SV_query_from = SV_query_to
updated_SV_query_to = SV_query_from
updated_SV_hit_from = SV_hit_to
updated_SV_hit_to = SV_hit_from
SV_genomepos_inalign = SV_genome_pos[(updated_SV_hit_from - 1):updated_SV_hit_to]
if SV_keyseq not in updated_SV_qseq.replace("-", "") or SV_keyseq not in updated_SV_hseq.replace("-", ""):
continue
SV_qseq, updated_SV_hseq, updated_SV_midline, key_qseq_in_align, key_hseq_in_align = adjust_1seg_indel_pos(seqid, updated_SV_qseq, updated_SV_hseq, updated_SV_midline, SV_genomepos_inalign, SV)
if "ND" in SV_qseq:
continue
if SV_keyseq == key_hseq_in_align == key_qseq_in_align:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[:-1])
SV_read_num[f"exact_{output_qual_pair}"][unique_seqidi] = 1
SV_read_ids["exact"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
else:
for seqidi in all_seqids:
for output_qual_pair in output_qual_pairs:
seqidi_qual_cutoff, seqidi_perc_cutoff = output_qual_pair.split("_")
if all_seqids[seqidi][seqidi_qual_cutoff] > float(seqidi_perc_cutoff):
unique_seqidi = "_".join(seqidi.split("_")[:-1])
SV_read_num[f"fuzzy_{output_qual_pair}"][unique_seqidi] = 1
SV_read_ids["fuzzy"].append([seqidi, str(all_seqids[seqidi]["20"]), str(all_seqids[seqidi]["30"])])
exact_SV_read_ids = SV_read_ids["exact"]
fuzzy_SV_read_ids = SV_read_ids["fuzzy"]
exact_SV_read_num = [len(SV_read_num[f"exact_{op}"]) for op in output_qual_pairs]
fuzzy_SV_read_num = [(len(SV_read_num[f"fuzzy_{op}"]) + len(SV_read_num[f"exact_{op}"])) for op in output_qual_pairs]
return exact_SV_read_ids, fuzzy_SV_read_ids, exact_SV_read_num, fuzzy_SV_read_num
def process_sv(line, centromere_regions, output_qual_pair_SV_num, out_SV_fh, out_SV_reads_fh, qual_perc_cutoff, clean_deepseq_folder, output_qual_pairs):
files_to_delete = []
lst = line.strip().split("\t")
initial_SV = lst[0]