def main():
usage = 'usage: %prog [options] <feature gff/bed>'
parser = OptionParser(usage)
parser.add_option('-g', dest='gff_file', help='Filter the TEs by overlap with genes in the given gff file [Default: %default]')
parser.add_option('-r', dest='repeats_gff', default='%s/research/common/data/genomes/hg19/annotation/repeatmasker/hg19.fa.out.tp.gff' % home_dir)
parser.add_option('-n', dest='null_iterations', type=int, default=50, help='Number of shuffles to perform to estimate null distribution [Default: %default]')
(options,args) = parser.parse_args()
if len(args) != 1:
parser.error('Must provide a gff file for the feature of interest.')
else:
feature_gff = args[0]
############################################
# GFF filter
############################################
# filter TEs and features by gff file
if options.gff_file:
# filter TE GFF
te_gff_fd, te_gff_file = tempfile.mkstemp()
subprocess.call('intersectBed -a %s -b %s > %s' % (options.repeats_gff, options.gff_file, te_gff_file), shell=True)
options.repeats_gff = te_gff_file
# filter feature GFF
feature_gff_gff_fd, feature_gff_gff_file = tempfile.mkstemp()
subprocess.call('intersectBed -s -u -f 0.5 -a %s -b %s > %s' % (feature_gff, options.gff_file, feature_gff_gff_file), shell=True)
feature_gff = feature_gff_gff_file
############################################
# lengths
############################################
# compute size of search space
if options.gff_file:
genome_length = count_gff(options.gff_file)
else:
genome_length = count_hg19()
# compute feature length
feature_len, feature_num = feature_stats(feature_gff)
if feature_num == 0:
print >> sys.stderr, 'Zero features'
exit()
# hash counted repeat genomic bp
te_in = open(options.repeats_gff)
genome_te_bp = hash_te(te_in)
te_in.close()
############################################
# convert feature gff to bed
############################################
if feature_gff[-3:] == 'gtf':
feature_bed_fd, feature_bed_file = tempfile.mkstemp()
subprocess.call('gtf2bed.py %s > %s' % (feature_gff,feature_bed_file), shell=True)
elif feature_gff[-3:] == 'gff':
feature_bed_fd, feature_bed_file = tempfile.mkstemp()
subprocess.call('gff2bed.py %s > %s' % (feature_gff,feature_bed_file), shell=True)
elif feature_gff[-3:] == 'bed':
feature_bed_file = feature_gff
else:
parser.error('Cannot recognize gff format suffix')
############################################
# null distribution
############################################
shuffle_bed_fd, shuffle_bed_file = tempfile.mkstemp()
te_null_bp = {}
for ni in range(options.null_iterations):
print >> sys.stderr, ni
# shuffle feature bed
subprocess.call('shuffleBed -i %s -g %s/research/common/data/genomes/hg19/assembly/human.hg19.genome -excl %s/research/common/data/genomes/hg19/assembly/hg19_gaps.bed > %s' % (feature_bed_file, home_dir, home_dir, shuffle_bed_file), shell=True)
# intersect w/ TEs and hash overlaps
te_tmp_bp = intersect_hash(options.repeats_gff, shuffle_bed_file)
for te in genome_te_bp:
te_null_bp.setdefault(te,[]).append(te_tmp_bp.get(te,0))
############################################
# actual
############################################
te_bp = intersect_hash(options.repeats_gff, feature_gff)
############################################
# compute stats and print
############################################
lines = []
p_vals = []
for te in genome_te_bp:
feature_freq = float(te_bp.get(te,0))/feature_len
genome_freq = float(genome_te_bp[te])/genome_length
fold_change = feature_freq / genome_freq
#print te, stats.mean(te_null_bp[te]), stats.sd(te_null_bp[te])
null_u, null_sd = stats.mean_sd(te_null_bp[te])
if null_sd == 0:
null_sd = 1.0
if fold_change > 1:
p = norm.sf(te_bp[te]-1, loc=null_u, scale=null_sd)
else:
p = norm.cdf(te_bp.get(te,0), loc=null_u, scale=null_sd)
p_vals.append(p)
cols = (te[0], te[1], te_bp.get(te,0), feature_freq, genome_freq, fold_change, p)
lines.append('%-18s %-18s %8d %11.2e %11.2e %9.2f %10.2e' % cols)
# correct for multiple hypotheses correction
q_vals = fdr.ben_hoch(p_vals)
for i in range(len(lines)):
qline = lines[i] + ' %10.2e' % q_vals[i]
print qline
############################################
# clean
############################################
os.close(shuffle_bed_fd)
os.remove(shuffle_bed_file)
if feature_gff[-3:] != 'bed':
os.close(feature_bed_fd)
os.remove(feature_bed_file)
if options.gff_file:
os.close(te_gff_fd)
os.remove(te_gff_file)
os.close(feature_gff_gff_fd)
os.remove(feature_gff_gff_file)
def main():
usage = 'usage: %prog [options] <bam> <ref_gtf>'
parser = OptionParser(usage)
# IO options
parser.add_option('-o',
dest='out_dir',
default='uniform',
help='Output directory [Default: %default]')
# window options
parser.add_option('-w',
dest='window_size',
type='int',
default=25,
help='Window size for counting [Default: %default]')
parser.add_option(
'-i',
'--ignore',
dest='ignore_gff',
help=
'Ignore reads overlapping overlapping troublesome regions in the given GFF file'
)
parser.add_option('-u',
'--unstranded',
dest='unstranded',
action='store_true',
default=False,
help='Sequencing is unstranded [Default: %default]')
# cufflinks options
parser.add_option(
'--cuff_done',
dest='cuff_done',
action='store_true',
default=False,
help=
'The Cufflinks run to estimate the model parameters is already done [Default: %default]'
)
parser.add_option('-t',
dest='threads',
type='int',
default=2,
help='Number of threads to use [Default: %default]')
# debug options
parser.add_option('-v',
'--verbose',
dest='verbose',
action='store_true',
default=False,
help='Verbose output [Default: %default]')
parser.add_option('-g',
'--gene',
dest='gene_only',
help='Call peaks on the specified gene only')
#parser.add_option('--print_windows', dest='print_windows', default=False, action='store_true', help='Print statistics for all windows [Default: %default]')
(options, args) = parser.parse_args()
if len(args) != 2:
parser.error(usage)
else:
bam = args[0]
ref_gtf = args[1]
clip_peaks.out_dir = options.out_dir
if not os.path.isdir(clip_peaks.out_dir):
os.mkdir(clip_peaks.out_dir)
############################################
# parameterize
############################################
if not options.cuff_done:
# make a new gtf w/ unspliced RNAs
update_ref_gtf = clip_peaks.prerna_gtf(ref_gtf)
subprocess.call(
'cufflinks -o %s -p %d -G %s %s' %
(clip_peaks.out_dir, options.threads, update_ref_gtf, bam),
shell=True)
# store transcripts
transcripts = clip_peaks.read_genes('%s/transcripts.gtf' %
clip_peaks.out_dir,
key_id='transcript_id')
# merge overlapping genes
g2t_merge, antisense_clusters = clip_peaks.merged_g2t(
'%s/transcripts.gtf' % clip_peaks.out_dir, options.unstranded)
if options.unstranded:
# alter strands
clip_peaks.ambiguate_strands(transcripts, g2t_merge,
antisense_clusters)
# set transcript FPKMs
clip_peaks.set_transcript_fpkms(transcripts,
clip_peaks.out_dir,
missing_fpkm=0)
# possibly limit genes to examine
if options.gene_only:
gene_ids = []
for gids in g2t_merge.keys():
if options.gene_only in gids.split(','):
gene_ids.append(gids)
if len(gene_ids) == 0:
print >> sys.stderr, 'gene_id %s not found' % options.gene_only
exit(1)
else:
gene_ids = g2t_merge.keys()
############################################
# filter BAM
############################################
if options.ignore_gff:
bam_ignore_fd, bam_ignore_file = tempfile.mkstemp(
dir='%s/research/scratch/temp' % os.environ['HOME'])
subprocess.call('intersectBed -v -abam %s -b %s > %s' %
(bam, options.ignore_gff, bam_ignore_file),
shell=True)
bam = bam_ignore_file
############################################
# process genes
############################################
# index
subprocess.call('samtools index %s' % bam, shell=True)
# initialize stats
table_out = open('%s/uniformity_table.txt' % clip_peaks.out_dir, 'w')
id_list = []
fpkm_list = []
# open bam
bam_in = pysam.Samfile(bam, 'rb')
# for each gene
for gene_id in gene_ids:
# make a more focused transcript hash for this gene
gene_transcripts = {}
for tid in g2t_merge[gene_id]:
gene_transcripts[tid] = transcripts[tid]
# obtain basic gene attributes
(gchrom, gstrand, gstart,
gend) = clip_peaks.gene_attrs(gene_transcripts)
# initialize window counts
transcript_isoform_counts = {}
for tid in gene_transcripts:
transcript_isoform_counts[tid] = []
# choose a single event position and weight the reads
read_pos_weights = clip_peaks.position_reads(bam_in,
gchrom,
gstart,
gend,
gstrand,
mapq_zero=True)
# process read alignments
for (pos, weight, mm) in read_pos_weights:
# map pos to isoforms
iso_pos = {}
for tid in gene_transcripts:
iso_pos[tid] = isoform_position(gene_transcripts[tid], pos)
# sum fpkms for hit isoforms
fpkm_sum = sum([
gene_transcripts[tid].fpkm for tid in gene_transcripts
if iso_pos[tid] != None
])
if fpkm_sum <= 0:
pass
#print >> sys.stderr, 'No FPKM for %s at %d' % (gene_id,pos)
else:
# distribute read to isoform counts
for tid in gene_transcripts:
if iso_pos[tid] != None:
win_i = int(iso_pos[tid] / options.window_size)
while win_i >= len(transcript_isoform_counts[tid]):
transcript_isoform_counts[tid].append(0)
transcript_isoform_counts[tid][
win_i] += weight * gene_transcripts[
tid].fpkm / fpkm_sum
# compute window stats
for tid in gene_transcripts:
if gene_transcripts[tid].fpkm > 1 and len(
transcript_isoform_counts[tid]) > 5:
u, sd = stats.mean_sd(transcript_isoform_counts[tid][:-1])
if u > 0:
id_list.append(sd * sd / u)
fpkm_list.append(gene_transcripts[tid].fpkm)
cols = (tid, gene_transcripts[tid].fpkm,
len(transcript_isoform_counts[tid]) - 1, u, sd,
id_list[-1])
print >> table_out, '%-20s %8.2f %6d %7.2f %7.2f %5.3f' % cols
bam_in.close()
table_out.close()
############################################
# summary stats
############################################
median = stats.median(id_list)
mean = stats.mean(id_list)
fpkm_cv_sum = sum([id_list[i] * fpkm_list[i] for i in range(len(id_list))])
fpkm_sum = sum(fpkm_list)
fpkm_mean = fpkm_cv_sum / fpkm_sum
logfpkm_cv_sum = sum([
id_list[i] * math.log(fpkm_list[i] + 1, 2) for i in range(len(id_list))
])
logfpkm_sum = sum([math.log(f + 1, 2) for f in fpkm_list])
logfpkm_mean = logfpkm_cv_sum / logfpkm_sum
# print
print 'Median: %7.4f' % median
print 'Mean: %7.4f' % mean
print 'FPKM-weighted mean: %7.4f' % fpkm_mean
print 'logFPKM-weighted mean: %7.4f' % logfpkm_mean
# clean cufflinks output
if not options.cuff_done:
os.remove(update_ref_gtf)
os.remove('%s/skipped.gtf' % clip_peaks.out_dir)
os.remove('%s/genes.fpkm_tracking' % clip_peaks.out_dir)
if options.ignore_gff:
os.close(bam_ignore_fd)
os.remove(bam_ignore_file)
def main():
usage = "usage: %prog [options] <input_file>"
parser = OptionParser(usage)
parser.add_option(
"-k",
dest="k_fold",
type="int",
default=10,
help="Number of folds to use for cross-validation [Default: %default]",
)
parser.add_option(
"--lambda_min",
dest="lambda_min",
type="float",
default=0.01,
help="Minimum -lambda value to attempt [Default: %default]",
)
parser.add_option(
"--lambda_max",
dest="lambda_max",
type="float",
default=10.0,
help="Maximum -lambda value to attempt [Default: %default]",
)
parser.add_option(
"--lambda_mult",
dest="lambda_mult",
type="float",
default=2.0,
help="Multiplier for next -lambda value to attempt [Default: %default]",
)
parser.add_option(
"-l",
dest="lesser_kmers",
action="store_true",
default=False,
help="Use all kmers of length less than and equal to that given by -k [Default: %default]",
)
# parser.add_option('-m', dest='model_file', help='File to output model to')
parser.add_option(
"-p", dest="parallel", type="int", default=4, help="Number of parallel threads to run [Default: %default]"
)
parser.add_option(
"-r",
dest="replicates",
type="int",
default=1,
help="Number of times to repeat the optimization for each fold [Default: %default]",
)
parser.add_option(
"-w", dest="weights", action="store_true", default=False, help="Print a summary of the weight vectors"
)
(options, args) = parser.parse_args()
if len(args) != 1:
parser.error("Must provide input file")
else:
input_file = args[0]
input_base = os.path.splitext(input_file)[0]
if options.weights:
summarize_weights(input_base, options)
exit()
# determine % of positive examples
input_pos, input_total = positive_percent(input_file)
f1_base = input_pos / float(input_total) # trust me, it works
for r in range(options.replicates):
rep_dir = "%s_rep%d" % (input_base, r)
if os.path.isdir(rep_dir):
shutil.rmtree(rep_dir)
os.mkdir(rep_dir)
os.chdir(rep_dir)
# divide data into folds
divide_data("../" + input_file, options.k_fold)
# collect pegasos commands
cmds = []
peg_lambda = options.lambda_min
while peg_lambda <= options.lambda_max:
# run on each fold
for f in range(options.k_fold):
cmds.append(
"pegasos -lambda %f -modelFile fold%d/train_%.1e.mod fold%d/train.dat &> /dev/null"
% (peg_lambda, f, peg_lambda, f)
)
# increase lambda
peg_lambda *= options.lambda_mult
# exceute pegasos commands
util.exec_par(cmds, options.parallel)
# start to clean up space
for f in range(options.k_fold):
os.remove("fold%d/train.dat" % f)
os.chdir("..")
# collect results
peg_lambda = options.lambda_min
while peg_lambda <= options.lambda_max:
recalls = []
precisions = []
failed = False
for r in range(options.replicates):
if not failed:
outcomes = {"tp": 0, "fp": 0, "fn": 0}
# collect each fold
for f in range(options.k_fold):
if not compute_accuracy(outcomes, "%s_rep%d/fold%d" % (input_base, r, f), peg_lambda):
failed = True
break
# save
if not failed:
recalls.append(float(outcomes["tp"]) / (outcomes["tp"] + outcomes["fn"]))
precisions.append(float(outcomes["tp"]) / (outcomes["tp"] + outcomes["fp"]))
# summarize and print
if failed:
print "%.1e %8s %7s %8s %7s %8s %8s" % (peg_lambda, "NA", "NA", "NA", "NA", "NA", "NA")
else:
recall, rsd = stats.mean_sd(recalls)
rsd /= math.sqrt(len(recalls))
precision, psd = stats.mean_sd(precisions)
psd /= math.sqrt(len(precisions))
# null_p = 1.0-binom.cdf(int(recall*input_total+0.5)-1, int(recall*input_total/precision + 0.5), float(input_pos)/input_total)
f1 = 2 * recall * precision / (recall + precision)
# print '%.1e %8.3f %6.3f %8.3f %6.3f %8.3f %8.3f %8.1e' % (peg_lambda, recall, rsd, precision, psd, f1, (f1-f1_base), null_p)
print "%.1e %8.4f %7.4f %8.4f %7.4f %8.4f %8.4f" % (
peg_lambda,
recall,
rsd,
precision,
psd,
f1,
(f1 - f1_base),
)
peg_lambda *= options.lambda_mult
def main():
usage = 'usage: %prog [options] <bam> <ref_gtf>'
parser = OptionParser(usage)
# IO options
parser.add_option('-o', dest='out_dir', default='uniform', help='Output directory [Default: %default]')
# window options
parser.add_option('-w', dest='window_size', type='int', default=25, help='Window size for counting [Default: %default]')
parser.add_option('-i', '--ignore', dest='ignore_gff', help='Ignore reads overlapping overlapping troublesome regions in the given GFF file')
parser.add_option('-u', '--unstranded', dest='unstranded', action='store_true', default=False, help='Sequencing is unstranded [Default: %default]')
# cufflinks options
parser.add_option('--cuff_done', dest='cuff_done', action='store_true', default=False, help='The Cufflinks run to estimate the model parameters is already done [Default: %default]')
parser.add_option('-t', dest='threads', type='int', default=2, help='Number of threads to use [Default: %default]')
# debug options
parser.add_option('-v', '--verbose', dest='verbose', action='store_true', default=False, help='Verbose output [Default: %default]')
parser.add_option('-g', '--gene', dest='gene_only', help='Call peaks on the specified gene only')
#parser.add_option('--print_windows', dest='print_windows', default=False, action='store_true', help='Print statistics for all windows [Default: %default]')
(options,args) = parser.parse_args()
if len(args) != 2:
parser.error(usage)
else:
bam = args[0]
ref_gtf = args[1]
clip_peaks.out_dir = options.out_dir
if not os.path.isdir(clip_peaks.out_dir):
os.mkdir(clip_peaks.out_dir)
############################################
# parameterize
############################################
if not options.cuff_done:
# make a new gtf w/ unspliced RNAs
update_ref_gtf = clip_peaks.prerna_gtf(ref_gtf)
subprocess.call('cufflinks -o %s -p %d -G %s %s' % (clip_peaks.out_dir, options.threads, update_ref_gtf, bam), shell=True)
# store transcripts
transcripts = clip_peaks.read_genes('%s/transcripts.gtf'%clip_peaks.out_dir, key_id='transcript_id')
# merge overlapping genes
g2t_merge, antisense_clusters = clip_peaks.merged_g2t('%s/transcripts.gtf'%clip_peaks.out_dir, options.unstranded)
if options.unstranded:
# alter strands
clip_peaks.ambiguate_strands(transcripts, g2t_merge, antisense_clusters)
# set transcript FPKMs
clip_peaks.set_transcript_fpkms(transcripts, clip_peaks.out_dir, missing_fpkm=0)
# possibly limit genes to examine
if options.gene_only:
gene_ids = []
for gids in g2t_merge.keys():
if options.gene_only in gids.split(','):
gene_ids.append(gids)
if len(gene_ids) == 0:
print >> sys.stderr, 'gene_id %s not found' % options.gene_only
exit(1)
else:
gene_ids = g2t_merge.keys()
############################################
# filter BAM
############################################
if options.ignore_gff:
bam_ignore_fd, bam_ignore_file = tempfile.mkstemp(dir='%s/research/scratch/temp' % os.environ['HOME'])
subprocess.call('intersectBed -v -abam %s -b %s > %s' % (bam, options.ignore_gff, bam_ignore_file), shell=True)
bam = bam_ignore_file
############################################
# process genes
############################################
# index
subprocess.call('samtools index %s' % bam, shell=True)
# initialize stats
table_out = open('%s/uniformity_table.txt' % clip_peaks.out_dir, 'w')
id_list = []
fpkm_list = []
# open bam
bam_in = pysam.Samfile(bam, 'rb')
# for each gene
for gene_id in gene_ids:
# make a more focused transcript hash for this gene
gene_transcripts = {}
for tid in g2t_merge[gene_id]:
gene_transcripts[tid] = transcripts[tid]
# obtain basic gene attributes
(gchrom, gstrand, gstart, gend) = clip_peaks.gene_attrs(gene_transcripts)
# initialize window counts
transcript_isoform_counts = {}
for tid in gene_transcripts:
transcript_isoform_counts[tid] = []
# choose a single event position and weight the reads
read_pos_weights = clip_peaks.position_reads(bam_in, gchrom, gstart, gend, gstrand, mapq_zero=True)
# process read alignments
for (pos, weight, mm) in read_pos_weights:
# map pos to isoforms
iso_pos = {}
for tid in gene_transcripts:
iso_pos[tid] = isoform_position(gene_transcripts[tid], pos)
# sum fpkms for hit isoforms
fpkm_sum = sum([gene_transcripts[tid].fpkm for tid in gene_transcripts if iso_pos[tid] != None])
if fpkm_sum <= 0:
pass
#print >> sys.stderr, 'No FPKM for %s at %d' % (gene_id,pos)
else:
# distribute read to isoform counts
for tid in gene_transcripts:
if iso_pos[tid] != None:
win_i = int(iso_pos[tid] / options.window_size)
while win_i >= len(transcript_isoform_counts[tid]):
transcript_isoform_counts[tid].append(0)
transcript_isoform_counts[tid][win_i] += weight*gene_transcripts[tid].fpkm/fpkm_sum
# compute window stats
for tid in gene_transcripts:
if gene_transcripts[tid].fpkm > 1 and len(transcript_isoform_counts[tid]) > 5:
u, sd = stats.mean_sd(transcript_isoform_counts[tid][:-1])
if u > 0:
id_list.append(sd*sd/u)
fpkm_list.append(gene_transcripts[tid].fpkm)
cols = (tid, gene_transcripts[tid].fpkm, len(transcript_isoform_counts[tid])-1, u, sd, id_list[-1])
print >> table_out, '%-20s %8.2f %6d %7.2f %7.2f %5.3f' % cols
bam_in.close()
table_out.close()
############################################
# summary stats
############################################
median = stats.median(id_list)
mean = stats.mean(id_list)
fpkm_cv_sum = sum([id_list[i]*fpkm_list[i] for i in range(len(id_list))])
fpkm_sum = sum(fpkm_list)
fpkm_mean = fpkm_cv_sum / fpkm_sum
logfpkm_cv_sum = sum([id_list[i]*math.log(fpkm_list[i]+1,2) for i in range(len(id_list))])
logfpkm_sum = sum([math.log(f+1,2) for f in fpkm_list])
logfpkm_mean = logfpkm_cv_sum / logfpkm_sum
# print
print 'Median: %7.4f' % median
print 'Mean: %7.4f' % mean
print 'FPKM-weighted mean: %7.4f' % fpkm_mean
print 'logFPKM-weighted mean: %7.4f' % logfpkm_mean
# clean cufflinks output
if not options.cuff_done:
os.remove(update_ref_gtf)
os.remove('%s/skipped.gtf' % clip_peaks.out_dir)
os.remove('%s/genes.fpkm_tracking' % clip_peaks.out_dir)
if options.ignore_gff:
os.close(bam_ignore_fd)
os.remove(bam_ignore_file)