def __init__(self, fastq):
# Store name
self.fastq = fastq
# Basename and extension
self.basename = strip_ngs_extensions(self.fastq)
self.extension = self.fastq[len(self.basename):]
self.basename = os.path.basename(self.basename)
# Values that should be derived from the name
# (should be set by subclass)
self.sample_name = None
self.sample_number = None
self.barcode_sequence = None
self.lane_number = None
self.read_number = None
self.set_number = None
self.is_index_read = False
def fastq_strand_output(fastq):
"""
Generate name for fastq_strand.py output
Given a Fastq file name, the output from fastq_strand.py
will look like:
- {FASTQ}_fastq_strand.txt
Arguments:
fastq (str): name of Fastq file
Returns:
tuple: fastq_strand.py output (without leading paths)
"""
return "%s_fastq_strand.txt" % strip_ngs_extensions(
os.path.basename(fastq))
def fastqc_output(fastq):
"""
Generate name of FastQC outputs
Given a Fastq file name, the outputs from FastQC will look
like:
- {FASTQ}_fastqc/
- {FASTQ}_fastqc.html
- {FASTQ}_fastqc.zip
Arguments:
fastq (str): name of Fastq file
Returns:
tuple: FastQC outputs (without leading paths)
"""
base_name = "%s_fastqc" % strip_ngs_extensions(os.path.basename(fastq))
return (base_name, base_name + '.html', base_name + '.zip')
def fastqc_output(fastq):
"""
Generate name of FastQC outputs
Given a Fastq file name, the outputs from FastQC will look
like:
- {FASTQ}_fastqc/
- {FASTQ}_fastqc.html
- {FASTQ}_fastqc.zip
Arguments:
fastq (str): name of Fastq file
Returns:
tuple: FastQC outputs (without leading paths)
"""
base_name = "%s_fastqc" % strip_ngs_extensions(os.path.basename(fastq))
return (base_name,base_name+'.html',base_name+'.zip')
def fastq_screen_output(fastq, screen_name):
"""
Generate name of fastq_screen output files
Given a Fastq file name and a screen name, the outputs from
fastq_screen will look like:
- {FASTQ}_{SCREEN_NAME}_screen.png
- {FASTQ}_{SCREEN_NAME}_screen.txt
Arguments:
fastq (str): name of Fastq file
screen_name (str): name of screen
Returns:
tuple: fastq_screen output names (without leading path)
"""
base_name = "%s_%s_screen" % (strip_ngs_extensions(
os.path.basename(fastq)), str(screen_name))
return (base_name + '.png', base_name + '.txt')
def fastq_screen_output(fastq,screen_name):
"""
Generate name of fastq_screen output files
Given a Fastq file name and a screen name, the outputs from
fastq_screen will look like:
- {FASTQ}_{SCREEN_NAME}_screen.png
- {FASTQ}_{SCREEN_NAME}_screen.txt
Arguments:
fastq (str): name of Fastq file
screen_name (str): name of screen
Returns:
tuple: fastq_screen output names (without leading path)
"""
base_name = "%s_%s_screen" % (strip_ngs_extensions(os.path.basename(fastq)),
str(screen_name))
return (base_name+'.png',base_name+'.txt')
def fastq_strand(argv, working_dir=None):
"""
Driver for fastq_strand
Generate strandedness statistics for single FASTQ or
FASTQ pair, by running STAR using one or more genome
indexes
"""
# Process command line
p = argparse.ArgumentParser(
description="Generate strandedness statistics "
"for FASTQ or FASTQpair, by running STAR using "
"one or more genome indexes",
version=__version__)
p.add_argument("r1", metavar="READ1", default=None, help="R1 Fastq file")
p.add_argument("r2",
metavar="READ2",
default=None,
nargs="?",
help="R2 Fastq file")
p.add_argument("-g",
"--genome",
dest="star_genomedirs",
metavar="GENOMEDIR",
default=None,
action="append",
help="path to directory with STAR index "
"for genome to use (use as an alternative "
"to -c/--conf; can be specified multiple "
"times to include additional genomes)")
p.add_argument("--subset",
type=int,
default=10000,
help="use a random subset of read pairs "
"from the input Fastqs; set to zero to "
"use all reads (default: 10000)")
p.add_argument("-o",
"--outdir",
default=None,
help="specify directory to write final "
"outputs to (default: current directory)")
p.add_argument("-c",
"--conf",
metavar="FILE",
default=None,
help="specify delimited 'conf' file with "
"list of NAME and STAR index directory "
"pairs. NB if a conf file is supplied "
"then any indices specifed on the command "
"line will be ignored")
p.add_argument("-n",
type=int,
default=1,
help="number of threads to run STAR with "
"(default: 1)")
p.add_argument("--counts",
action="store_true",
help="include the count sums for "
"unstranded, 1st read strand aligned and "
"2nd read strand aligned in the output "
"file (default: only include percentages)")
p.add_argument("--keep-star-output",
action="store_true",
help="keep the output from STAR (default: "
"delete outputs on completion)")
args = p.parse_args(argv)
# Print parameters
print("READ1\t: %s" % args.r1)
print("READ2\t: %s" % args.r2)
# Check that STAR is on the path
star_exe = find_program("STAR")
if star_exe is None:
logging.critical("STAR not found")
return 1
print("STAR\t: %s" % star_exe)
# Gather genome indices
genome_names = {}
if args.conf is not None:
print("Conf file\t: %s" % args.conf)
star_genomedirs = []
with open(args.conf, 'r') as fp:
for line in fp:
if line.startswith('#'):
continue
name, star_genomedir = line.rstrip().split('\t')
star_genomedirs.append(star_genomedir)
# Store an associated name
genome_names[star_genomedir] = name
else:
star_genomedirs = args.star_genomedirs
if not star_genomedirs:
logging.critical("No genome indices specified")
return 1
print("Genomes:")
for genome in star_genomedirs:
print("- %s" % genome)
# Output directory
if args.outdir is None:
outdir = os.getcwd()
else:
outdir = os.path.abspath(args.outdir)
if not os.path.exists(outdir):
logging.critical("Output directory doesn't exist: %s" % outdir)
return 1
# Output file
outfile = "%s_fastq_strand.txt" % os.path.join(
outdir, os.path.basename(strip_ngs_extensions(args.r1)))
if os.path.exists(outfile):
logging.warning("Removing existing output file '%s'" % outfile)
os.remove(outfile)
# Prefix for temporary output
prefix = "fastq_strand_"
# Working directory
if working_dir is None:
working_dir = os.getcwd()
else:
working_dir = os.path.abspath(working_dir)
if not os.path.isdir(working_dir):
raise Exception("Bad working directory: %s" % working_dir)
print("Working directory: %s" % working_dir)
# Make subset of input read pairs
nreads = sum(1 for i in getreads(os.path.abspath(args.r1)))
print("%d reads" % nreads)
if args.subset == 0:
print("Using all read pairs in Fastq files")
subset = nreads
elif args.subset > nreads:
print("Actual number of read pairs smaller than requested subset")
subset = nreads
else:
subset = args.subset
print("Using random subset of %d read pairs" % subset)
if subset == nreads:
subset_indices = [i for i in xrange(nreads)]
else:
subset_indices = random.sample(xrange(nreads), subset)
fqs_in = filter(lambda fq: fq is not None, (args.r1, args.r2))
fastqs = []
for fq in fqs_in:
fq_subset = os.path.join(working_dir, os.path.basename(fq))
if fq_subset.endswith(".gz"):
fq_subset = '.'.join(fq_subset.split('.')[:-1])
fq_subset = "%s.subset.fq" % '.'.join(fq_subset.split('.')[:-1])
with open(fq_subset, 'w') as fp:
for read in getreads_subset(os.path.abspath(fq), subset_indices):
fp.write('\n'.join(read) + '\n')
fastqs.append(fq_subset)
# Make directory to keep output from STAR
if args.keep_star_output:
star_output_dir = os.path.join(
outdir, "STAR.%s.outputs" %
os.path.basename(strip_ngs_extensions(args.r1)))
print("Output from STAR will be copied to %s" % star_output_dir)
# Check if directory already exists from earlier run
if os.path.exists(star_output_dir):
# Move out of the way
i = 0
backup_dir = "%s.bak" % star_output_dir
while os.path.exists(backup_dir):
i += 1
backup_dir = "%s.bak%s" % (star_output_dir, i)
logging.warning("Moving existing output directory to %s" %
backup_dir)
os.rename(star_output_dir, backup_dir)
# Make the directory
os.mkdir(star_output_dir)
# Write output to a temporary file
with tempfile.TemporaryFile() as fp:
# Iterate over genome indices
for star_genomedir in star_genomedirs:
# Basename for output for this genome
try:
name = genome_names[star_genomedir]
except KeyError:
name = star_genomedir
# Build a command line to run STAR
star_cmd = [star_exe]
star_cmd.extend([
'--runMode', 'alignReads', '--genomeLoad', 'NoSharedMemory',
'--genomeDir',
os.path.abspath(star_genomedir)
])
star_cmd.extend(['--readFilesIn', fastqs[0]])
if len(fastqs) > 1:
star_cmd.append(fastqs[1])
star_cmd.extend([
'--quantMode', 'GeneCounts', '--outSAMtype', 'BAM', 'Unsorted',
'--outSAMstrandField', 'intronMotif', '--outFileNamePrefix',
prefix, '--runThreadN',
str(args.n)
])
print("Running %s" % ' '.join(star_cmd))
try:
subprocess.check_output(star_cmd, cwd=working_dir)
except subprocess.CalledProcessError as ex:
raise Exception("STAR returned non-zero exit code: %s" %
ex.returncode)
# Save the outputs
if args.keep_star_output:
# Make a subdirectory for this genome index
genome_dir = os.path.join(star_output_dir,
name.replace(os.sep, "_"))
print("Copying STAR outputs to %s" % genome_dir)
os.mkdir(genome_dir)
for f in os.listdir(working_dir):
if f.startswith(prefix):
shutil.copy(os.path.join(working_dir, f),
os.path.join(genome_dir, f))
# Process the STAR output
star_tab_file = os.path.join(working_dir,
"%sReadsPerGene.out.tab" % prefix)
if not os.path.exists(star_tab_file):
raise Exception("Failed to find .out file: %s" % star_tab_file)
sum_col2 = 0
sum_col3 = 0
sum_col4 = 0
with open(star_tab_file) as out:
for i, line in enumerate(out):
if i < 4:
# Skip first four lines
continue
# Process remaining delimited columns
cols = line.rstrip('\n').split('\t')
sum_col2 += int(cols[1])
sum_col3 += int(cols[2])
sum_col4 += int(cols[3])
print("Sums:")
print("- col2: %d" % sum_col2)
print("- col3: %d" % sum_col3)
print("- col4: %d" % sum_col4)
if sum_col2 > 0.0:
forward_1st = float(sum_col3) / float(sum_col2) * 100.0
reverse_2nd = float(sum_col4) / float(sum_col2) * 100.0
else:
logging.warning("Sum of mapped reads is zero!")
forward_1st = 0.0
reverse_2nd = 0.0
print("Strand percentages:")
print("- 1st forward: %.2f%%" % forward_1st)
print("- 2nd reverse: %.2f%%" % reverse_2nd)
# Append to output file
data = [name, "%.2f" % forward_1st, "%.2f" % reverse_2nd]
if args.counts:
data.extend([sum_col2, sum_col3, sum_col4])
fp.write("%s\n" % "\t".join([str(d) for d in data]))
# Finished iterating over genomes
# Rewind temporary output file
fp.seek(0)
with open(outfile, 'w') as out:
# Header
out.write("#fastq_strand version: %s\t"
"#Aligner: %s\t"
"#Reads in subset: %s\n" % (__version__, "STAR", subset))
columns = ["Genome", "1st forward", "2nd reverse"]
if args.counts:
columns.extend([
"Unstranded", "1st read strand aligned",
"2nd read strand aligned"
])
out.write("#%s\n" % "\t".join(columns))
# Copy content from temp to final file
for line in fp:
out.write(line)
return 0
def fastq_strand(argv,working_dir=None):
"""
Driver for fastq_strand
Generate strandedness statistics for single FASTQ or
FASTQ pair, by running STAR using one or more genome
indexes
"""
# Process command line
p = argparse.ArgumentParser(
description="Generate strandedness statistics "
"for FASTQ or FASTQpair, by running STAR using "
"one or more genome indexes",
version=__version__)
p.add_argument("r1",metavar="READ1",
default=None,
help="R1 Fastq file")
p.add_argument("r2",metavar="READ2",
default=None,
nargs="?",
help="R2 Fastq file")
p.add_argument("-g","--genome",
dest="star_genomedirs",metavar="GENOMEDIR",
default=None,
action="append",
help="path to directory with STAR index "
"for genome to use (use as an alternative "
"to -c/--conf; can be specified multiple "
"times to include additional genomes)")
p.add_argument("--subset",
type=int,
default=10000,
help="use a random subset of read pairs "
"from the input Fastqs; set to zero to "
"use all reads (default: 10000)")
p.add_argument("-o","--outdir",
default=None,
help="specify directory to write final "
"outputs to (default: current directory)")
p.add_argument("-c","--conf",metavar="FILE",
default=None,
help="specify delimited 'conf' file with "
"list of NAME and STAR index directory "
"pairs. NB if a conf file is supplied "
"then any indices specifed on the command "
"line will be ignored")
p.add_argument("-n",
type=int,
default=1,
help="number of threads to run STAR with "
"(default: 1)")
p.add_argument("--counts",
action="store_true",
help="include the count sums for "
"unstranded, 1st read strand aligned and "
"2nd read strand aligned in the output "
"file (default: only include percentages)")
p.add_argument("--keep-star-output",
action="store_true",
help="keep the output from STAR (default: "
"delete outputs on completion)")
args = p.parse_args(argv)
# Print parameters
print "READ1\t: %s" % args.r1
print "READ2\t: %s" % args.r2
# Check that STAR is on the path
star_exe = find_program("STAR")
if star_exe is None:
logging.critical("STAR not found")
return 1
print "STAR\t: %s" % star_exe
# Gather genome indices
genome_names = {}
if args.conf is not None:
print "Conf file\t: %s" % args.conf
star_genomedirs = []
with open(args.conf,'r') as fp:
for line in fp:
if line.startswith('#'):
continue
name,star_genomedir = line.rstrip().split('\t')
star_genomedirs.append(star_genomedir)
# Store an associated name
genome_names[star_genomedir] = name
else:
star_genomedirs = args.star_genomedirs
if not star_genomedirs:
logging.critical("No genome indices specified")
return 1
print "Genomes:"
for genome in star_genomedirs:
print "- %s" % genome
# Output directory
if args.outdir is None:
outdir = os.getcwd()
else:
outdir = os.path.abspath(args.outdir)
if not os.path.exists(outdir):
logging.critical("Output directory doesn't exist: %s" %
outdir)
return 1
# Output file
outfile = "%s_fastq_strand.txt" % os.path.join(
outdir,
os.path.basename(strip_ngs_extensions(args.r1)))
if os.path.exists(outfile):
logging.warning("Removing existing output file '%s'" % outfile)
os.remove(outfile)
# Prefix for temporary output
prefix = "fastq_strand_"
# Working directory
if working_dir is None:
working_dir = os.getcwd()
else:
working_dir = os.path.abspath(working_dir)
if not os.path.isdir(working_dir):
raise Exception("Bad working directory: %s" % working_dir)
print "Working directory: %s" % working_dir
# Make subset of input read pairs
nreads = sum(1 for i in getreads(os.path.abspath(args.r1)))
print "%d reads" % nreads
if args.subset == 0:
print "Using all read pairs in Fastq files"
subset = nreads
elif args.subset > nreads:
print "Actual number of read pairs smaller than requested subset"
subset = nreads
else:
subset = args.subset
print "Using random subset of %d read pairs" % subset
if subset == nreads:
subset_indices = [i for i in xrange(nreads)]
else:
subset_indices = random.sample(xrange(nreads),subset)
fqs_in = filter(lambda fq: fq is not None,(args.r1,args.r2))
fastqs = []
for fq in fqs_in:
fq_subset = os.path.join(working_dir,
os.path.basename(fq))
if fq_subset.endswith(".gz"):
fq_subset = '.'.join(fq_subset.split('.')[:-1])
fq_subset = "%s.subset.fq" % '.'.join(fq_subset.split('.')[:-1])
with open(fq_subset,'w') as fp:
for read in getreads_subset(os.path.abspath(fq),
subset_indices):
fp.write('\n'.join(read) + '\n')
fastqs.append(fq_subset)
# Make directory to keep output from STAR
if args.keep_star_output:
star_output_dir = os.path.join(outdir,
"STAR.%s.outputs" %
os.path.basename(
strip_ngs_extensions(args.r1)))
print "Output from STAR will be copied to %s" % star_output_dir
# Check if directory already exists from earlier run
if os.path.exists(star_output_dir):
# Move out of the way
i = 0
backup_dir = "%s.bak" % star_output_dir
while os.path.exists(backup_dir):
i += 1
backup_dir = "%s.bak%s" % (star_output_dir,i)
logging.warning("Moving existing output directory to %s" %
backup_dir)
os.rename(star_output_dir,backup_dir)
# Make the directory
os.mkdir(star_output_dir)
# Write output to a temporary file
with tempfile.TemporaryFile() as fp:
# Iterate over genome indices
for star_genomedir in star_genomedirs:
# Basename for output for this genome
try:
name = genome_names[star_genomedir]
except KeyError:
name = star_genomedir
# Build a command line to run STAR
star_cmd = [star_exe]
star_cmd.extend([
'--runMode','alignReads',
'--genomeLoad','NoSharedMemory',
'--genomeDir',os.path.abspath(star_genomedir)])
star_cmd.extend(['--readFilesIn',
fastqs[0]])
if len(fastqs) > 1:
star_cmd.append(fastqs[1])
star_cmd.extend([
'--quantMode','GeneCounts',
'--outSAMtype','BAM','Unsorted',
'--outSAMstrandField','intronMotif',
'--outFileNamePrefix',prefix,
'--runThreadN',str(args.n)])
print "Running %s" % ' '.join(star_cmd)
try:
subprocess.check_output(star_cmd,cwd=working_dir)
except subprocess.CalledProcessError as ex:
raise Exception("STAR returned non-zero exit code: %s" %
ex.returncode)
# Save the outputs
if args.keep_star_output:
# Make a subdirectory for this genome index
genome_dir = os.path.join(star_output_dir,
name.replace(os.sep,"_"))
print "Copying STAR outputs to %s" % genome_dir
os.mkdir(genome_dir)
for f in os.listdir(working_dir):
if f.startswith(prefix):
shutil.copy(os.path.join(working_dir,f),
os.path.join(genome_dir,f))
# Process the STAR output
star_tab_file = os.path.join(working_dir,
"%sReadsPerGene.out.tab" % prefix)
if not os.path.exists(star_tab_file):
raise Exception("Failed to find .out file: %s" % star_tab_file)
sum_col2 = 0
sum_col3 = 0
sum_col4 = 0
with open(star_tab_file) as out:
for i,line in enumerate(out):
if i < 4:
# Skip first four lines
continue
# Process remaining delimited columns
cols = line.rstrip('\n').split('\t')
sum_col2 += int(cols[1])
sum_col3 += int(cols[2])
sum_col4 += int(cols[3])
print "Sums:"
print "- col2: %d" % sum_col2
print "- col3: %d" % sum_col3
print "- col4: %d" % sum_col4
if sum_col2 > 0.0:
forward_1st = float(sum_col3)/float(sum_col2)*100.0
reverse_2nd = float(sum_col4)/float(sum_col2)*100.0
else:
logging.warning("Sum of mapped reads is zero!")
forward_1st = 0.0
reverse_2nd = 0.0
print "Strand percentages:"
print "- 1st forward: %.2f%%" % forward_1st
print "- 2nd reverse: %.2f%%" % reverse_2nd
# Append to output file
data = [name,
"%.2f" % forward_1st,
"%.2f" % reverse_2nd]
if args.counts:
data.extend([sum_col2,sum_col3,sum_col4])
fp.write("%s\n" % "\t".join([str(d) for d in data]))
# Finished iterating over genomes
# Rewind temporary output file
fp.seek(0)
with open(outfile,'w') as out:
# Header
out.write("#fastq_strand version: %s\t"
"#Aligner: %s\t"
"#Reads in subset: %s\n" % (__version__,
"STAR",
subset))
columns = ["Genome","1st forward","2nd reverse"]
if args.counts:
columns.extend(["Unstranded",
"1st read strand aligned",
"2nd read strand aligned"])
out.write("#%s\n" % "\t".join(columns))
# Copy content from temp to final file
for line in fp:
out.write(line)
return 0
