def extract_marker_gene_reads(bams,out_prefix,cores):
N = min(len(bams),cores,cpu_count())
extract_reads.extract_reads(bamlist=bams,cores=N,prefix=out_prefix)
# merge single reads
cmd = ['cat']
for i in xrange(N):
hs = '%s.%d.single.fastq' % (out_prefix,i)
if file_size(hs)>0:
cmd.append(hs)
if len(cmd)>1:
f = open(out_prefix+'.single.fastq','w')
subprocess.call(cmd,stdout=f)
f.close()
# merge pe1 reads
cmd = ['cat']
for i in xrange(N):
h1 = '%s.%d.pe1.fastq' % (out_prefix,i)
if file_size(h1)>0:
cmd.append(h1)
if len(cmd)>1:
f = open(out_prefix+'.pe1.fastq','w')
subprocess.call(cmd,stdout=f)
f.close()
# merge pe2 reads
cmd = ['cat']
for i in xrange(N):
h2 = '%s.%d.pe2.fastq' % (out_prefix,i)
if file_size(h2)>0:
cmd.append(h2)
if len(cmd)>1:
f = open(out_prefix+'.pe2.fastq','w')
subprocess.call(cmd,stdout=f)
f.close()
# remove temporary file
cmd = ['rm']
for i in xrange(N):
hs = '%s.%d.single.fastq' % (out_prefix,i)
h1 = '%s.%d.pe1.fastq' % (out_prefix,i)
h2 = '%s.%d.pe2.fastq' % (out_prefix,i)
if os.path.exists(hs):
cmd.append(hs)
if os.path.exists(h1):
cmd.append(h1)
if os.path.exists(h2):
cmd.append(h2)
subprocess.call(cmd)
def extract_bam_reads(bam, verbose=False):
'''
It is to extract short reads from the input bam file.
'''
if verbose:
logging.info('extract short reads from ' + os.path.basename(bam))
prefix = os.path.join(os.getcwd(),os.path.splitext(os.path.basename(bam))[0])
PE1,PE2,SIN = extract_reads.extract_reads(bamlist=[bam],prefix=prefix)
# merge
os.system('cat {} {} {} | seqtk seq -A > {}'.format(PE1,PE2,SIN,prefix+'.fasta'))
# remove temporary file
cmd = ['rm','-f',PE1,PE2,SIN]
call(cmd)
return prefix+'.fasta'
def extract_bam_reads(bam, verbose=False):
'''
It is to extract short reads from the input bam file.
'''
if verbose:
logging.info('extract short reads from ' + os.path.basename(bam))
prefix = os.path.join(os.getcwd(),os.path.splitext(os.path.basename(bam))[0])
PE1,PE2,SIN = extract_reads.extract_reads(bamlist=[bam],prefix=prefix)
# merge
os.system('cat {} {} {} | seqtk seq -A > {}'.format(PE1,PE2,SIN,prefix+'.fasta'))
# remove temporary file
cmd = ['rm','-f',PE1,PE2,SIN]
call(cmd)
return prefix+'.fasta'
def extract_sample_gene_read(params):
smpl, bam = params
extract_reads.extract_reads(bamlist=[bam], prefix=smpl, cores=1)
return None
def extract_marker_gene_reads(bams, out_prefix, cores):
N = min(len(bams), cores, cpu_count())
extract_reads.extract_reads(bamlist=bams, cores=N, prefix=out_prefix)
def main():
# required packages
import os
import argparse
import datetime
import time
parser = argparse.ArgumentParser(
description='Main script of the FUCHS pipeline.'
'For a detailed help see https://github.com/dieterich-lab/FUCHS '
'or the included README.rst file.')
# input
parser.add_argument(
'-C',
'--circIDs',
dest='circlefile',
default='none',
help='Tab-separated file chr:start_end(tab)read1,read2,read3.')
parser.add_argument(
'-D',
'--DCC',
dest='CircRNACount',
default='none',
help=
'If you mapped with STAR and are using step1 you need to provide a list'
' of circle ids (CircRNACount or CircCoordinates from DCC)'
'You must supply either -C or -DCC')
parser.add_argument(
'-J',
'--chimericJunctions',
dest='chimeric_junction',
default='none',
help=
'If you mapped with STAR and are using step1 you need to provide the paired end Chimeric.junction.out file here'
)
parser.add_argument(
'-F',
'--mate1',
dest='mate1',
default='none',
help=
'If you mapped with STAR and are using step1 you need to provide the mate1.Chimeric.junction.out file here (optional if ends were mapped separately)'
)
parser.add_argument(
'-R',
'--mate2',
dest='mate2',
default='none',
help=
'If you mapped with STAR and are using step1 you need to provide the mate2.Chimeric.junction.out file here (optional if ends were mapped separately)'
)
parser.add_argument(
'-B',
'--bamfile',
dest='bamfile',
required=True,
help=
'BAM file containing chimeric reads, linear reads may be in it but are not required.'
)
parser.add_argument('-A',
'--annotation',
dest='bedfile',
required=True,
help='bed formatted feature file including exons.')
# output
parser.add_argument(
'-O',
'--outFolder',
dest='out_folder',
default='.',
help=
'Output folder. There will be a sub folder for the sample containing a BAM file '
'for each circle.')
parser.add_argument('-N',
'--sampleName',
dest='sample',
required=True,
help='sample name to title every thing.')
# options
parser.add_argument(
'-r',
'--thresholdReads',
dest='reads',
default=5,
type=int,
help='Circle has to have at least <r> reads to be analysed.')
# TODO: default: no multi map
parser.add_argument(
'-q',
'--thresholdMapq',
dest='mapq',
default=3,
type=int,
help=
'MAPQ cutoff, only reads passing this threshold will be written to circle BAM file.'
)
# TODO: add 0 based info
parser.add_argument('-c',
'--splitCharacter',
dest='split_character',
default='_',
help='feature name separator.')
parser.add_argument(
'-e',
'--exonIndex',
dest='exon_index',
default=3,
type=int,
help=
'Field indicating the exon number after splitting feature name by split_character (for the annotation file).'
)
parser.add_argument(
'-p',
'--annotationFormat',
dest='ref_platform',
default='refseq',
help=
'Specifies the annotation platform which was used (refseq or ensembl)')
parser.add_argument(
'-s',
'--skipSteps',
dest='skipped_steps',
default='none',
help=
'Comma separated list of steps that should be skipped (e.g. step3,step4,step6)'
)
parser.add_argument(
'-T',
'--tmp',
dest='tmp_folder',
default='/tmp/',
help='Folder to store temporary files generated by pybedtools.')
parser.add_argument('-P',
'--cpus',
dest='num_cpus',
default=4,
type=int,
help='Number of CPUs used.')
args = parser.parse_args()
# parse arguments
circles = os.path.expanduser(args.circlefile)
circle_ids = os.path.expanduser(args.CircRNACount)
paired = os.path.expanduser(
args.chimeric_junction) # not the greatest naming scheme
mate1 = os.path.expanduser(args.mate1)
mate2 = os.path.expanduser(args.mate2)
bamfile = os.path.expanduser(args.bamfile)
bedfile = os.path.expanduser(args.bedfile)
outfolder = os.path.expanduser(args.out_folder) + '/'
sample = args.sample
num_cpus = args.num_cpus
cutoff_reads = args.reads
cutoff_mapq = args.mapq
exon_index = args.exon_index
split_character = args.split_character
platform = args.ref_platform
skipped_steps = args.skipped_steps.split(',')
tmp_folder = os.path.expanduser(args.tmp_folder) + '/'
# start writing down FUCHS time for retracing
print('Started FUCHS at %s' % (datetime.datetime.now()))
dt = str(datetime.datetime.now())
start_time = time.time()
# make log file
# TODO
# test if command line was correct
if circles == 'none' and circle_ids == 'none':
print(
'ERROR, you need to specify either a -C or -DCC.\nIf you mapped and detected your circRNAs with STAR/DCC you may indicate \n-DCC CircRNACount, -CJ Chimeric.junction.out, -m1 mate1.Chimeric.junction.out and -m2 mate2.Chimeric.junction.out\nif you used a different program, please supply a circID list using -C.\n'
)
quit()
if not circles == 'none' and not circle_ids == 'none':
print(
'You have indicated both -C and -DCC, this is not necessary, we will skip step1 (read extraction from the STAR output) and proceed with the circID file\n'
)
circle_ids == 'none'
skipped_steps += ['step1']
if not circle_ids == 'none' and paired == 'none':
print(
'You have indicated that you detected your circRNAs using STAR/DCC with the -DCC flag, \nhowever you did not specify a Chimeric.junction.out file, this is necessary, \nplease specify at least -CJ, if you have paired end data also specify -m1/-m2\n'
)
quit()
# convert relative paths names to absolute path names
working_dir = os.getcwd()
if not circles == 'none' and not os.path.isabs(circles):
circles = os.path.abspath(os.path.join(os.getcwd(), circles))
print('changed circID file to %s\n' % (circles))
if not circles == 'none' and not os.path.exists(circles):
print('ERROR, no such file or directory: %s' % (circles))
quit()
if not circle_ids == 'none' and not os.path.isabs(circle_ids):
circle_ids = os.path.abspath(os.path.join(os.getcwd(), circle_ids))
print('changed CircRNACount file to %s\n' % (circle_ids))
if not circle_ids == 'none' and not os.path.exists(circle_ids):
print('ERROR, no such file or directory: %s' % (circle_ids))
quit()
if not paired == 'none' and not os.path.isabs(paired):
paired = os.path.abspath(os.path.join(os.getcwd(), paired))
print('changed Chimeric.junction.out file to %s\n' % (paired))
if not paired == 'none' and not os.path.exists(paired):
print('ERROR, no such file or directory: %s' % (paired))
quit()
if not mate2 == 'none' and not os.path.isabs(mate2):
mate2 = os.path.abspath(os.path.join(os.getcwd(), mate2))
print('changed mate2.Chimeric.junction.out file to %s\n' % (mate2))
if not mate2 == 'none' and not os.path.exists(mate2):
print('ERROR, no such file or directory: %s' % (mate2))
quit()
if not mate1 == 'none' and not os.path.isabs(mate1):
mate1 = os.path.abspath(os.path.join(os.getcwd(), mate1))
print('changed mate1.Chimeric.junction.out file to %s\n' % (mate1))
if not mate1 == 'none' and not os.path.exists(mate1):
print('ERROR, no such file or directory: %s' % (mate1))
quit()
if not os.path.isabs(bamfile):
bamfile = os.path.abspath(os.path.join(os.getcwd(), bamfile))
print('changed bamfile file to %s\n' % (bamfile))
if not os.path.exists(bamfile):
print('ERROR, no such file or directory: %s' % (bamfile))
quit()
if not os.path.isabs(outfolder):
outfolder = os.path.abspath(os.path.join(os.getcwd(), outfolder))
print('changed output folder to %s\n' % (outfolder))
if not os.path.isdir(outfolder):
os.mkdir(outfolder)
if not os.path.isabs(tmp_folder):
tmp_folder = os.path.abspath(os.path.join(os.getcwd(), tmp_folder))
print('changed tmp folder to %s\n' % (tmp_folder))
if not os.path.isdir(tmp_folder):
os.mkdir(tmp_folder)
if not os.path.isabs(bedfile):
bedfile = os.path.abspath(os.path.join(os.getcwd(), bedfile))
print('changed bedfile file to %s\n' % (bedfile))
if not os.path.exists(bedfile):
print('ERROR, no such file or directory: %s' % (bedfile))
quit()
accepted_platforms = ('refseq', 'ensembl')
platform = platform.lower()
if not platform in accepted_platforms:
print(
'ERROR please specify an accepted annotation platform. Possible options are: refseq or ensembl'
)
quit()
print "The following analysis steps will be skipped: " + '%s' % ', '.join(
map(str, skipped_steps))
# Step 1: (optional) if DCC was used, extract circle read names from junction file
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'w')
output_file.write('FUCHS is starting at %s\n\n' % (dt))
output_file.write(
'%s: starting to get readnames from Chimeric.junction.out\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step1' in skipped_steps:
circles = "%s.reads.txt" % paired
if not os.path.isfile(circles):
import get_readnames_from_DCC as get_readnames
names = get_readnames.get_readnames_from_DCC(
circle_ids, paired, mate1, mate2)
names.run()
else:
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')),
'a')
output_file.write(
'\tskipping get_readnames_from_DCC because %s exists already\n'
% (circles))
output_file.close()
# Step2 : extract circle reads from sample bam file
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write(
'\tfinished\n\n%s: starting to extract chimeric reads from bamfile\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step2' in skipped_steps:
import extract_reads as extract_reads
er = extract_reads.extract_reads(cutoff_reads, cutoff_mapq, circles,
bamfile, outfolder, sample,
tmp_folder, num_cpus)
er.run()
# Step3 : (optional) get information about possibly rolling circles
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write(
'\tfinished\n\n%s: starting to get mate pair information\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step3' in skipped_steps:
if not os.path.isfile('%s/%s.mate_status.txt' % (outfolder, sample)):
import get_mate_information as mateinformation
mi = mateinformation.mate_information(platform, split_character,
bedfile, outfolder, sample,
tmp_folder, num_cpus)
mi.run()
else:
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')),
'a')
output_file.write(
'\tskipping get_mate_information because %s/%s.mate_status.txt exists already\n'
% (outfolder, sample))
output_file.close()
# Step4 : (optional) find exon skipping events
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write('\tfinished\n\n%s: starting to detect skipped exons\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step4' in skipped_steps:
if not os.path.isfile('%s/%s.skipped_exons.bed' % (outfolder, sample)):
import detect_skipped_exons as skipped_exons
se = skipped_exons.detect_skipped_exons(outfolder, sample, bedfile,
tmp_folder, platform,
num_cpus)
se.run()
else:
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')),
'a')
output_file.write(
'\tskipping detect_skipped_exons because %s/%s.skipped_exons.bed exists already\n'
% (outfolder, sample))
output_file.close()
# Step5 : (optional) identify different circles within the same host gene
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write(
'\tfinished\n\n%s: starting to detect alternative splicing\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step5' in skipped_steps:
if not os.path.isfile('%s/%s.alternative_splicing.txt' %
(outfolder, sample)):
import detect_splicing_variants as splicing_variants
sv = splicing_variants.detect_splicing_variants(
split_character, platform, circles, bedfile, outfolder, sample,
tmp_folder, num_cpus)
sv.run()
else:
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')),
'a')
output_file.write(
'\tskipping detect_splicing_variants because %s/%s.alternative_splicing.txt exists already\n'
% (outfolder, sample))
output_file.close()
# Step6 : (optional) generate coverage profile for each circle
# (one transcript per gene, best if most fitting transcript)
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write(
'\tfinished\n\n%s: starting to generate coverage profiles\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step6' in skipped_steps:
if not os.path.isfile('%s/%s.exon_counts.bed' %
(outfolder, sample)) and not os.path.isdir(
'%s/%s.coverage_profiles/' %
(outfolder, sample)):
import get_coverage_profile as coverage_profile
sv = coverage_profile.get_coverage_profile(exon_index,
split_character,
platform, bedfile,
outfolder, sample,
tmp_folder, num_cpus)
sv.run()
else:
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')),
'a')
output_file.write(
'\tskipping get_coverage_profile because %s/%s.exon_counts.bed exists already\n'
% (outfolder, sample))
output_file.close()
# Step7 : (optional, requires step 5)
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write(
'\tfinished\n\n%s: starting to summarize the coverage profiles\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step7' in skipped_steps:
if not os.path.isfile(
'%s/%s.coverage_profiles/coverage_profiles.all_circles.pdf' %
(outfolder, sample)):
if os.path.isdir('%s/%s.coverage_profiles/' % (outfolder, sample)):
os.system(
'summarized_coverage_profiles.R %s/%s.coverage_profiles' %
(outfolder, sample))
else:
output_file = open(
'%s/%s.logfile.%s' %
(outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write(
'\tYou are trying cluster the coverage profiles without '
'generating coverage profiles first, please run step 5 (get_coverage_profile)\n'
)
output_file.close()
else:
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')),
'a')
output_file.write(
'\tskipping summarized_coverage_profiles.R because %s/%s.coverage_profiles/coverage_profiles.all_circles.pdf already exists\n'
% (outfolder, sample))
output_file.close()
# Step8 : (optional, requires step6) pictures for all circles
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write(
'\tfinished\n\n%s: starting to visualize the coverage profiles\n' %
(datetime.datetime.now()))
output_file.close()
if not 'step8' in skipped_steps:
if os.path.isdir('%s/%s.coverage_profiles/' % (outfolder, sample)):
files = os.listdir('%s/%s.coverage_profiles' % (outfolder, sample))
folders = os.listdir(outfolder)
if not '%s.coverage_pictures' % (sample) in folders:
os.mkdir('%s/%s.coverage_pictures' % (outfolder, sample))
def run_r_parallel(f):
if f.endswith('.txt'):
os.system(
'make_coverage_picture.R %s/%s.coverage_profiles/%s %s/%s.coverage_pictures/'
% (outfolder, sample, f, outfolder, sample))
from pathos.multiprocessing import ProcessingPool as Pool
pool = Pool(num_cpus)
pool.map(run_r_parallel, files)
else:
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')),
'a')
output_file.write(
'\tYou are trying to generate coverage pictures '
'without generating coverage profiles, please run step 5 (get_coverage_profile)\n'
)
output_file.close()
output_file = open(
'%s/%s.logfile.%s' % (outfolder, sample, dt.replace(' ', '_')), 'a')
output_file.write('\tfinished\n\n\nFUCHS finished at %s\n\n' %
(datetime.datetime.now()))
output_file.write("FUCHS took --- %s minutes ---\n\n" % (round(
(time.time() - start_time) / 60.0)))
output_file.close()
def extract_marker_gene_reads(bams,out_prefix,cores):
N = min(len(bams),cores,cpu_count())
extract_reads.extract_reads(bamlist=bams,cores=N,prefix=out_prefix)
def extract_sample_gene_read(params):
smpl, bam = params
extract_reads.extract_reads(bamlist=[bam], prefix=smpl, cores=1)
return None
