def multi_to_fastq(sample_name, multi_bam, paired_end, alignment_file,
threads):
algn_parent = Path(alignment_file).parent
srt_multi_bam = Path(algn_parent, "_sorted.multi.bam")
out_SE = Path(algn_parent, sample_name + "_multimap.fastq")
out_R1 = Path(algn_parent, sample_name + "_multimap_R1.fastq")
out_R2 = Path(algn_parent, sample_name + "_multimap_R2.fastq")
if not paired_end:
print(f"Writing multi-mapped reads to {out_SE}...")
bamfile = BedTool(multi_bam)
BedTool.bam_to_fastq(bamfile, fq=out_SE)
else:
print("Sorting multi-mapped reads...")
pysam.sort("--threads", f"{threads}", "-n", "-o", f"{srt_multi_bam}",
f"{multi_bam}")
sorted_bamfile = BedTool(srt_multi_bam)
print(f"Writing multi-mapped reads to {out_R1} {out_R2}...")
BedTool.bam_to_fastq(sorted_bamfile, fq=out_R1, fq2=out_R2)
def remap(fq, genome_index_path, genome_index_name, snp_index_path, \
snp_index_name, threads, out_dir, snp_tolerance, keep_temp, mismatches):
# remapping of multi and unmapped reads from round 1
# generate fastq from unmaped reads
nomap = out_dir + fq.split("/")[-1].split(".")[0] + ".nomapping.bam"
nomap_bed = BedTool(nomap)
nomap_fastq = "".join(nomap.split(".bam")[:-1]) + ".fastq"
nomap_bed.bam_to_fastq(fq=nomap_fastq)
# generate fastq from multi-mapping reads
# first generate sam of primary alignments from multi-mappers
multi = out_dir + fq.split("/")[-1].split(".")[0] + ".unpaired_mult.bam"
cmd = "samtools view -@ " + str(
threads) + " -F 0x904 -o " + out_dir + "temp_multi.bam " + multi
subprocess.call(cmd, shell=True)
multi_bed = BedTool(out_dir + "temp_multi.bam")
multi_fastq = "".join(multi.split(".bam")[:-1]) + ".fastq"
multi_bed.bam_to_fastq(fq=multi_fastq)
os.remove(out_dir + "temp_multi.bam")
if not keep_temp:
os.remove(nomap)
os.remove(multi)
if not mismatches == None:
mismatch_string = "--max-mismatches " + str(mismatches) + " "
elif mismatches == None:
mismatch_string = ""
output_prefix = fq.split("/")[-1].split(".")[0] + "_remap"
if snp_tolerance:
map_cmd = "gsnap -D " + genome_index_path + " -d " + genome_index_name + " -V " + snp_index_path + " -v " \
+ snp_index_name + " -t " + str(threads) + " --split-output " + out_dir + output_prefix + \
" --format=sam --genome-unk-mismatch=0 --md-lowercase-snp --ignore-trim-in-filtering 1 --force-single-end " + mismatch_string +\
nomap_fastq + " " + multi_fastq + " &>> " + out_dir + "remap_align.log"
else:
map_cmd = "gsnap -D " + genome_index_path + " -d " + genome_index_name + " -t " + str(threads) + \
" --split-output " + out_dir + output_prefix + " --format=sam --genome-unk-mismatch=0 --md-lowercase-snp --ignore-trim-in-filtering 1 --force-single-end " + mismatch_string + " " + \
nomap_fastq + " " + multi_fastq + " &>> " + out_dir + "remap_align.log"
log.info(
"Realigning multi-mapped and unmapped reads to {} with updated SNP index..."
.format(genome_index_name))
subprocess.call(map_cmd, shell=True)
os.remove(multi_fastq)
os.remove(nomap_fastq)
# remove transloc sam output if no reads present (often the case)
cmd = "samtools view -c " + out_dir + output_prefix + ".unpaired_transloc"
readcount = int(subprocess.check_output(cmd, shell=True))
if readcount == 0:
os.remove(out_dir + output_prefix + ".unpaired_transloc")
# write mapping stats and compress to bam - remove mutlimapping and unmapped unless keep_temp = True
log.info("Compressing SAM files, sorting, and computing mapping stats...")
with open(out_dir + "mapping_stats.txt", "a") as stats_out:
align_pathlist = Path(out_dir).glob(output_prefix + "*")
for file in align_pathlist:
if re.search("mult",
file.name) and not re.search("bam", file.name):
cmd = "samtools view -@ " + str(
threads) + " -F 0x904 -c " + out_dir + file.name
multi_count = int(subprocess.check_output(cmd, shell=True))
if keep_temp:
cmd = "samtools view -@ " + str(
threads
) + " -bh -o " + out_dir + file.name + ".bam " + out_dir + file.name
subprocess.call(cmd, shell=True)
os.remove(out_dir + file.name)
else:
os.remove(out_dir + file.name)
elif re.search("uniq",
file.name) and not re.search("bam", file.name):
cmd = "samtools view -@ " + str(
threads
) + " -bh " + out_dir + file.name + " | samtools sort -@ " + str(
threads) + " -o " + out_dir + file.name + ".bam" + " - "
subprocess.call(cmd, shell=True)
os.remove(out_dir + file.name)
# merge 1st run bam and remapped bam
merged_bam = out_dir + fq.split("/")[-1].split(
".")[0] + ".unpaired_uniq_remapMerge.bam"
cmd = "samtools merge " + merged_bam + " " + out_dir + file.name + ".bam " + out_dir + fq.split(
"/")[-1].split(".")[0] + ".unpaired_uniq.bam"
subprocess.call(cmd, shell=True)
#os.remove(out_dir + file.name + ".bam")
cmd = "samtools view -@ " + str(threads) + " -c " + merged_bam
unique_count = int(subprocess.check_output(cmd, shell=True))
unique_bam = merged_bam
elif re.search("nomapping",
file.name) and not re.search("bam", file.name):
cmd = "samtools view -@ " + str(
threads) + " -c " + out_dir + file.name
unmapped_count = int(subprocess.check_output(cmd, shell=True))
if keep_temp:
cmd = "samtools view -@ " + str(
threads
) + " -bh -o " + out_dir + file.name + ".bam " + out_dir + file.name
subprocess.call(cmd, shell=True)
os.remove(out_dir + file.name)
else:
os.remove(out_dir + file.name)
total_count = unique_count + multi_count + unmapped_count
stats_out.write("{}\nUniquely mapped reads: {:d} ({:.0%}) \nMulti-mapping reads: {:d} ({:.0%}) \nUnmapped reads: {:d} ({:.0%}) \nTotal: {:d}\n\n"\
.format(fq.split("/")[-1], unique_count, (unique_count/total_count),multi_count, (multi_count/total_count), unmapped_count, (unmapped_count/total_count), total_count))
alignstats_dict = defaultdict(list)
type_list = ["Uniquely mapped", "Multi-mapped", "Unmapped"]
for i, count in enumerate([unique_count, multi_count, unmapped_count]):
alignstats_dict["Lib"].append(fq.split("/")[-1].split(".")[0])
alignstats_dict["Type"].append(type_list[i])
alignstats_dict["Count"].append(count)
return (unique_bam, unique_count, alignstats_dict)
