def reads_replace(bam_file, total_chosen_reads, seqer, flow_order, lib_key,
barcode, tag, out_dir, aligner, aligner_index, is_single):
bam = pysam.AlignmentFile(bam_file)
edit_bam_reads = {}
for read in bam.fetch():
read_name = read.query_name
if read_name in total_chosen_reads:
strand = getReadStrand(read)
if read_name not in edit_bam_reads:
edit_bam_reads[read_name] = {}
if strand in total_chosen_reads[read_name]:
my_read = total_chosen_reads[read_name][strand]
read.query_sequence = my_read.query_sequence
read.query_qualities = my_read.query_qualities
if seqer == "life":
read = deal_life_reads(read, flow_order, lib_key, barcode)
if tag:
read = add_tag(read)
edit_bam_reads[read_name][strand] = read
else:
edit_bam_reads[read_name][strand] = read
# write edited reads into edit.bam
edit_bam_file = os.path.join(out_dir, "edit.bam")
edit_bam = pysam.AlignmentFile(edit_bam_file, 'wb', template=bam)
for read_name, readInfo in edit_bam_reads.items():
for strand, read in readInfo.items():
edit_bam.write(read)
edit_bam.close()
# write not edited reads into exclude.bam
exclude_bam_file = os.path.join(out_dir, "exclude.bam")
exclude_bam = pysam.AlignmentFile(exclude_bam_file, 'wb', template=bam)
for read in bam.fetch():
read_name = read.query_name
if read_name not in edit_bam_reads:
exclude_bam.write(read)
exclude_bam.close()
# remap the edited reads
header = os.path.join(out_dir, 'bam.header')
os.system('samtools view -H %s|grep "^@RG" > %s' % (bam_file, header))
head = open(header, 'r').readline().rstrip()
if not head:
head = None
edit_remap_bam_file = os.path.join(out_dir, "edit.remap.bam")
remap(aligner_index,
edit_bam_file,
edit_remap_bam_file,
aligner,
is_single,
header=head)
edit_remap_bam_sorted_prefix = os.path.join(out_dir, "edit.remap.sort")
edit_remap_bam_sorted_file = os.path.join(out_dir, "edit.remap.sort.bam")
bamSort(edit_remap_bam_file, edit_remap_bam_sorted_prefix)
bamIndex(edit_remap_bam_sorted_file)
if tag:
edit_remap_addtag_file = os.path.join(out_dir, "edit.remap.sort.bam")
bam_add_tag(edit_remap_bam_sorted_file, edit_remap_addtag_file)
else:
edit_remap_addtag_file = edit_remap_bam_sorted_file
return edit_remap_addtag_file, exclude_bam_file
def deal_haplotype(bam_file, haplotype, reffasta, haplotype_prefix, mindepth,
minmutreads, minmapq, diffcover, is_single,
is_multmapfilter, aligner, aligner_index, **kwargs):
reads_dict = OrderedDict()
bam = pysam.AlignmentFile(bam_file, 'rb')
reads = bam.fetch(reference=haplotype.chrom,
start=haplotype.start,
end=haplotype.end + 1)
depth = 0
for read in reads:
depth += 1
if read.reference_start is not None and not read.is_secondary and bin(
read.flag & 2048) != bin(2048):
if read.query_name not in reads_dict:
reads_dict[read.query_name] = {}
strand = getReadStrand(read)
reads_dict[read.query_name][strand] = read
# judge depth and mut reads whether qualified
if depth < int(mindepth):
print "depth less than min depth!"
return False, "haplotype in position %s:%s-%s: depth less than min depth(%s)" % (
haplotype.chrom, haplotype.start, haplotype.end, mindepth)
else:
mut_reads_num = int(depth * haplotype.freq)
if mut_reads_num < int(minmutreads):
print "mutation reads num less than minmutreads!"
return False, "haplotype in position %s:%s-%s: mut reads less than min mut reads(%s)" % (
haplotype.chrom, haplotype.start, haplotype.end, minmutreads)
print "start pick reads"
# print str(haplotype)
res = pick_reads(bam, reads_dict, mut_reads_num, is_single, minmapq,
is_multmapfilter)
if res[0] is False:
return False, "haplotype in position %s:%s-%s: %s" % (
haplotype.chrom, haplotype.start, haplotype.end, res[1])
chosen_reads, mate_reads = res
print "end pick reads"
# edit
my_chosen_reads = {}
my_mate_reads = {}
tmp_bam_file = haplotype_prefix + ".chosen.edited.bam"
tmp_bam = pysam.AlignmentFile(tmp_bam_file, 'wb', template=bam)
chosen_reads_num = 0
real_mut_reads_num = 0
for readName, readInfo in chosen_reads.items():
my_chosen_reads[readName] = {}
tmp_dict = {}
tmp_dict2 = {}
for strand, read in readInfo.items():
my_read = Read(read)
res = editRead(my_read, reffasta, haplotype.mutList)
if res is False:
continue
real_mut_reads_num += 1
sequence, quality, shift = res
read.query_sequence = sequence
read.query_qualities = quality
tmp_dict[strand] = my_read
tmp_dict2[strand] = read
if is_single:
for strand in tmp_dict:
my_chosen_reads[readName][strand] = tmp_dict[strand]
tmp_bam.write(tmp_dict2[strand])
chosen_reads_num += 1
else:
if len(tmp_dict) == 0:
continue
elif len(tmp_dict) == 1 and readName in mate_reads:
for strand in tmp_dict:
my_chosen_reads[readName][strand] = tmp_dict[strand]
tmp_bam.write(tmp_dict2[strand])
chosen_reads_num += 1
mate_read = mate_reads[readName]
my_mate_reads[readName] = Read(mate_read)
tmp_bam.write(mate_read)
elif len(tmp_dict) == 2:
for strand in tmp_dict:
my_chosen_reads[readName][strand] = tmp_dict[strand]
tmp_bam.write(tmp_dict2[strand])
chosen_reads_num += 1
tmp_bam.close()
# alignment and judge coverdiff whether qualified
chosen_bam_file = haplotype_prefix + ".chosen.remap.bam"
genome_index = aligner_index
remap(genome_index, tmp_bam_file, chosen_bam_file, aligner, is_single)
chosen_bam = pysam.AlignmentFile(chosen_bam_file)
if judge_coverdiff(bam, depth, chosen_bam, chosen_reads_num, haplotype,
float(diffcover)):
return my_chosen_reads, my_mate_reads, real_mut_reads_num, depth
else:
return False, "haplotype in position %s:%s-%s: coverdiff is less than minDiffCover" % (
haplotype.chrom, haplotype.start, haplotype.end)
def merge_edit_bam(bam_file, out_dir, is_single, total_modify_reads_file,
total_add_reads_file, used_bam_file, total_modify_list,
total_add_list, seqer, aligner, aligner_index, flow_order,
lib_key, barcode, tag):
bam = pysam.AlignmentFile(bam_file, 'rb')
edit_bam_file = os.path.join(out_dir, "edit.bam")
edit_bam = pysam.AlignmentFile(edit_bam_file, 'wb', template=bam)
readname_convert_file = os.path.join(out_dir, "readname_convert.txt")
fout_convert = open(readname_convert_file, 'w')
# edit_bam_reads = {}
used_bam = pysam.AlignmentFile(used_bam_file, 'rb')
used_reads = {}
for read in used_bam.fetch():
keyname = getKeyName(read)
used_reads[keyname] = read
used_bam.close()
# modify_read_name_dict = get_newname_dict(total_modify_list)
# add_read_name_dict = get_newname_dict(total_add_list)
modify_reads_seq, modify_reads_quan = get_sequence_dict(
total_modify_reads_file)
add_reads_seq, add_reads_quan = get_sequence_dict(total_add_reads_file)
if is_single:
with open(total_modify_list) as fin:
for line in fin:
if not line:
break
data = line.strip().split(",")
read1_name = data[0]
if read1_name not in used_reads:
continue
orig_read1 = used_reads[read1_name]
new_read1 = copy.deepcopy(orig_read1)
new_read1.query_sequence = modify_reads_seq[read1_name]
new_read1.query_qualities = modify_reads_quan[read1_name]
new_name = get_new_readname()
new_read1.query_name = new_name
if seqer == "life":
new_read1 = deal_life_reads(new_read1, flow_order, lib_key,
barcode)
fout_convert.write(
"%s: %s, %s, %s-%s\n" %
(new_name, orig_read1.query_name, new_read1.is_read1,
new_read1.reference_start, new_read1.reference_end))
edit_bam.write(new_read1)
fin.close()
with open(total_add_list) as fin:
for line in fin:
if not line:
break
data = line.strip().split(",")
read1_name = data[0]
if read1_name not in used_reads:
continue
orig_read1 = used_reads[read1_name]
new_read1 = copy.deepcopy(orig_read1)
new_read1.query_sequence = add_reads_seq[read1_name]
new_read1.query_qualities = add_reads_quan[read1_name]
new_name = get_new_readname()
new_read1.query_name = new_name
fout_convert.write(
"%s: %s, %s, %s-%s\n" %
(new_name, orig_read1.query_name, new_read1.is_read1,
new_read1.reference_start, new_read1.reference_end))
edit_bam.write(new_read1)
fin.close()
else:
with open(total_modify_list) as fin:
for line in fin:
if not line:
break
data = line.strip().split(",")
read1_name, read2_name = data[0], data[1]
if read1_name not in used_reads or read2_name not in used_reads:
continue
orig_read1 = used_reads[read1_name]
orig_read2 = used_reads[read2_name]
new_read1 = copy.deepcopy(orig_read1)
new_read2 = copy.deepcopy(orig_read2)
print read1_name
new_read1.query_sequence = modify_reads_seq[read1_name]
new_read1.query_qualities = modify_reads_quan[read1_name]
new_read2.query_sequence = modify_reads_seq[read2_name]
new_read2.query_qualities = modify_reads_quan[read2_name]
new_name = get_new_readname()
new_read1.query_name = new_name
new_read2.query_name = new_name
fout_convert.write(
"%s: %s, %s, %s-%s\n" %
(new_name, orig_read1.query_name, new_read1.is_read1,
new_read1.reference_start, new_read1.reference_end))
fout_convert.write(
"%s: %s, %s, %s-%s\n" %
(new_name, orig_read2.query_name, new_read2.is_read1,
new_read2.reference_start, new_read2.reference_end))
edit_bam.write(new_read1)
edit_bam.write(new_read2)
fin.close()
with open(total_add_list) as fin:
for line in fin:
if not line:
break
data = line.strip().split(",")
read1_name, read2_name = data[0], data[1]
if read1_name not in used_reads or read2_name not in used_reads:
continue
orig_read1 = used_reads[read1_name]
orig_read2 = used_reads[read2_name]
new_read1 = copy.deepcopy(orig_read1)
new_read2 = copy.deepcopy(orig_read2)
new_read1.query_sequence = add_reads_seq[read1_name]
new_read1.query_qualities = add_reads_quan[read1_name]
new_read2.query_sequence = add_reads_seq[read2_name]
new_read2.query_qualities = add_reads_quan[read2_name]
new_name = get_new_readname()
new_read1.query_name = new_name
new_read2.query_name = new_name
fout_convert.write(
"%s: %s, %s, %s-%s\n" %
(new_name, orig_read1.query_name, new_read1.is_read1,
new_read1.reference_start, new_read1.reference_end))
fout_convert.write(
"%s: %s, %s, %s-%s\n" %
(new_name, orig_read2.query_name, new_read2.is_read1,
new_read2.reference_start, new_read2.reference_end))
edit_bam.write(new_read1)
edit_bam.write(new_read2)
fin.close()
edit_bam.close()
header = os.path.join(out_dir, 'bam.header')
os.system('samtools view -H %s|grep "^@RG" > %s' % (bam_file, header))
head = open(header, 'r').readline().rstrip().replace('\t', '\\t')
if not head:
head = None
edit_remap_bam_file = os.path.join(out_dir, "edit.remap.bam")
remap(aligner_index, edit_bam_file, edit_remap_bam_file, aligner,
is_single, head)
edit_remap_bam_sorted_prefix = os.path.join(out_dir, "edit.remap.sort")
edit_remap_bam_sorted_file = os.path.join(out_dir, "edit.remap.sort.bam")
bamSort(edit_remap_bam_file, edit_remap_bam_sorted_prefix)
return edit_remap_bam_sorted_file