def rmdup(self):
msg = "Removing duplicates in %s" % self.bamfn
print(msg)
tempfn_stem = os.path.join(self.basedir, temp_filename())
pysam.rmdup("-s", self.bamfn, tempfn_stem)
tempfn_glob = glob.glob(tempfn_stem + '*')
assert len(tempfn_glob) == 1, "Unexpected number of temporary output files: %r" % tempfn_glob
tempfn = tempfn_glob[0]
# rename our dedupped bamfn
os.rename(tempfn, self.bamfn)
def proc_sam(arg):
samfile = arg[0]
rmdup = arg[1]
#se = arg[2]
print samfile
print rmdup
sam_dir = "/".join(samfile.split("/")[:-1]) + "/"
sam_prefix = os.path.basename(samfile).split(".sam")[0]
mapped_sam = sam_dir + sam_prefix + "_mapped.sam"
rmdup_sam = sam_dir + sam_prefix + "_rmdup.sam"
sort_sam = sam_dir + sam_prefix + "_sort"
if not os.path.exists(mapped_sam):
print "Removing unmapped..."
sam = pysam.Samfile(samfile, 'r')
mb = pysam.Samfile(mapped_sam, 'w', template=sam)
for read in sam:
if not read.is_unmapped:
mb.write(read)
mb.close()
print "Finished removing unmapped."
if not os.path.exists(rmdup_sam) and rmdup == "True":
print "Removing duplicates..."
pysam.rmdup("-S", mapped_sam, rmdup_sam)
os.remove(mapped_sam)
print "Sorting..."
pysam.sort(rmdup_sam, sort_sam)
os.remove(rmdup_sam)
else:
print "Sorting..."
pysam.sort(mapped_sam, sort_sam)
os.remove(mapped_sam)
print "Indexing..."
sort_sam = sort_sam + ".sam"
pysam.index(sort_sam)
samfile_fs = open(samfile + "_stat", 'w')
for line in pysam.flagstat(samfile):
samfile_fs.write(line)
samfile_fs.close
sort_sam_fs = open(sort_sam + "_stat", 'w')
for line in pysam.flagstat(sort_sam):
sort_sam_fs.write(line)
sort_sam_fs.close()
def proc_sam(arg):
samfile = arg[0]
rmdup = arg[1]
#se = arg[2]
print samfile
print rmdup
sam_dir = "/".join(samfile.split("/")[:-1]) + "/"
sam_prefix = os.path.basename(samfile).split(".sam")[0]
mapped_sam = sam_dir + sam_prefix + "_mapped.sam"
rmdup_sam = sam_dir + sam_prefix + "_rmdup.sam"
sort_sam = sam_dir + sam_prefix + "_sort"
if not os.path.exists(mapped_sam):
print "Removing unmapped..."
sam = pysam.Samfile(samfile, 'r')
mb = pysam.Samfile(mapped_sam, 'w', template=sam)
for read in sam:
if not read.is_unmapped:
mb.write(read)
mb.close()
print "Finished removing unmapped."
if not os.path.exists(rmdup_sam) and rmdup == "True":
print "Removing duplicates..."
pysam.rmdup("-S", mapped_sam, rmdup_sam)
os.remove(mapped_sam)
print "Sorting..."
pysam.sort(rmdup_sam, sort_sam)
os.remove(rmdup_sam)
else:
print "Sorting..."
pysam.sort(mapped_sam, sort_sam)
os.remove(mapped_sam)
print "Indexing..."
sort_sam = sort_sam + ".sam"
pysam.index(sort_sam)
samfile_fs = open(samfile + "_stat", 'w')
for line in pysam.flagstat(samfile):
samfile_fs.write(line)
samfile_fs.close
sort_sam_fs = open(sort_sam + "_stat", 'w')
for line in pysam.flagstat(sort_sam):
sort_sam_fs.write(line)
sort_sam_fs.close()
def trim_reads(bamfile):
"""
Wrapper to remove PCR duplicate reads from bed file
Input
bamfile -- location of bamfile on disk
assumes .bam ending of bam file
returns bamfile_trimed.bam file
"""
if not os.path.exists(bamfile):
raise NameError("file %s does not exist" % (bamfile))
outfile = ".".join(bamfile.split(".")[:-1])
outfile += ".rmdup.bam"
rmdup("-S", bamfile, outfile)
return outfile
def trim_reads(bamfile):
"""
Wrapper to remove PCR duplicate reads from bed file
Input
bamfile -- location of bamfile on disk
assumes .bam ending of bam file
returns bamfile_trimed.bam file
"""
if not os.path.exists(bamfile):
raise NameError("file %s does not exist" % (bamfile))
outfile = ".".join(bamfile.split(".")[:-1])
outfile += ".rmdup.bam"
rmdup("-S", bamfile, outfile)
return outfile
def rm_dup(self, inbam, outbam):
''' remove pcr duplicates '''
pysam.rmdup(inbam, outbam)
return
def rm_dup(self,inbam,outbam): ''' remove pcr duplicates ''' pysam.rmdup(inbam,outbam) return
def filter_main(fastq1,
fastq2,
bwa_index,
mapq,
outdir,
prefix,
threads,
to_file=False):
sys.stdout = logger.Logger(outdir + "/" + prefix + ".feather.log")
print(time.ctime() + " starting mapping and filtering operation")
check_arguments(fastq1, fastq2, bwa_index, mapq, threads)
paired_filename, bwa1_filename, bwa2_filename, bwa1_sorted_filename, bwa2_sorted_filename, combined_bwa_filename, qc_filename = set_filenames(
fastq1, fastq2, outdir, prefix)
#running bwa mem
for fastq, bwa_filename in [(fastq1, bwa1_filename),
(fastq2, bwa2_filename)]:
if fastq.endswith(".fastq") or fastq.endswith("fastq.gz"):
bwa_mem(fastq, bwa_index, threads, bwa_filename)
elif not (fastq.endswith(".sam") or fastq.endswith(".bam")):
exit(
"Error: Input file for filtering should be of type fastq, fastq.gz, sam, or bam. Exiting!"
)
if bwa1_filename.endswith(".bam"):
proc = subprocess.Popen("samtools view " + bwa1_filename +
" | awk ' $1 !~ /@/ {print $1}' " +
"| uniq -c|wc -l",
stdout=subprocess.PIPE,
shell=True)
read_count = proc.stdout.read().decode("utf-8")
else:
proc = subprocess.Popen("awk ' $1 !~ /@/ {print $1}' " +
bwa1_filename + "| uniq -c|wc -l",
stdout=subprocess.PIPE,
shell=True)
read_count = proc.stdout.read().decode("utf-8")
#pairing and filtering alignments for chimeric reads
for bwa_filename, bwa_sorted_filename in ([
bwa1_filename, bwa1_sorted_filename
], [bwa2_filename, bwa2_sorted_filename]):
bwa = pysam.AlignmentFile(bwa_filename)
if not is_sorted_queryname(bwa.header):
print(time.ctime() + " calling samtools sort for " + bwa_filename +
" storing in " + bwa_sorted_filename)
pysam.sort("-o", bwa_sorted_filename, "-n", "-@", str(threads),
bwa_filename)
else:
copyfile(bwa_filename, bwa_sorted_filename)
print(time.ctime() + " merging " + bwa1_sorted_filename + " and " +
bwa2_sorted_filename)
pysam.merge("-n", "-f", combined_bwa_filename, bwa1_sorted_filename,
bwa2_sorted_filename)
print(time.ctime() + " filtering and pairing reads")
filter_pair_reads(combined_bwa_filename, mapq, paired_filename,
qc_filename)
print(time.ctime() + " paired bam file generated. Sorting by coordinates.")
pysam.sort("-o", paired_filename + ".srt.bam", "-@", str(threads),
paired_filename + ".bam")
print(time.ctime() + " calling samtools rmdup")
pysam.rmdup(paired_filename + ".srt.bam", paired_filename + ".rmdup.bam")
#proc = subprocess.Popen(["samtools", "rmdup", paired_filename + ".srt.bam", paired_filename + ".rmdup.bam"])
#proc.communicate()
print(time.ctime() + " calling samtools flagstat on mapped file")
proc = subprocess.Popen("samtools flagstat " + paired_filename +
".srt.bam > " + paired_filename +
".srt.bam.flagstat",
shell=True)
proc.communicate()
with open(paired_filename + ".srt.bam.flagstat") as flag_file:
lines = flag_file.readlines()
uniquely_mapped_count = lines[7].split()[0]
print(time.ctime() +
" calling samtools flagstat on mapped and duplicate-removed file")
proc = subprocess.Popen("samtools flagstat " + paired_filename +
".rmdup.bam > " + paired_filename +
".rmdup.flagstat",
shell=True)
proc.communicate()
with open(paired_filename + ".rmdup.flagstat") as flag_file:
lines = flag_file.readlines()
duprmd_count = lines[7].split()[0]
intra_count = lines[11].split()[0]
intra_count = str(int(float(intra_count)) / 2)
print(time.ctime() + " calling samtools sort for sorting by query names")
#pysam.sort("-n", "-o", bwa_filename + ".srtn.rmdup.bam", paired_filename + ".rmdup.bam")
pysam.sort("-o", paired_filename + ".srtn.rmdup.bam", "-@", str(threads),
"-n", paired_filename + ".rmdup.bam")
#proc.communicate()
#proc.wait()
print(time.ctime() + " finishing filtering")
qc_filename = outdir + "/" + prefix + ".feather.qc"
with open(qc_filename, 'w') as outfile:
outfile.write("{0:70} {1}".format("number of sequencing pairs",
str(read_count)))
outfile.write("{0:70} {1} ".format(
"number of unqiuely mapped pairs (MAPQ >= " + str(mapq) + ")",
str(uniquely_mapped_count)))
outfile.write("\t({0:.2f}%)\n".format(
100 *
(int(float(uniquely_mapped_count)) / int(float(read_count)))))
outfile.write("{0:70} {1} ".format(
"number of pairs after duplicate removal", str(duprmd_count)))
outfile.write("\t({0:.2f}%)\n".format(
100 * (int(float(duprmd_count)) / int(float(read_count)))))
#outfile.write("{0:70} {1} ".format("number of interchromosomal pairs", str(intra_count)))
#outfile.write("\t({0:.2f}%)\n".format(100 * int(float(intra_count)) / int(float(read_count))))
return (paired_filename + ".srtn.rmdup.bam")