def add_promoter(genes_gff, chromsize, promoter_length):
"""This function adds a promoter of a certain length to each gene in the input file and exports an output pyBedTools object"""
# output = open(output_location, 'w') #make output file with write capability
# parse gff file containing only genes.
genes = BedTool(genes_gff)
# extract promoters upsteam using chromsize file and specified promoter length. r, no. of bp to add to end coordinate. s, based on strand.
promoters = genes.flank(g=chromsize, l=promoter_length, r=0, s=True)
return promoters
class GenomicSubset(object):
def __init__(self, name, path=paths.genome_subsets, assembly='hg19'):
self.assembly = assembly
self.name = name
self.bedtool = BedTool(path + name + '.bed').sort()
# Intersect the pathway with the appropriate genome build
# TODO: this step should be unnecessary if the pathways are correct
if name != self.assembly:
self.bedtool = GenomicSubset.reference_genome(
self.assembly).bedtool.intersect(self.bedtool).sort().saveas()
def expand_by(self, expansion_in_each_direction_Mb):
window_size_str = str(expansion_in_each_direction_Mb) + 'Mb'
print('total size before window addition:', self.bedtool.total_coverage(), 'bp')
# compute the flanks
# TODO: use 1cM instead of 1Mb
print('computing flanks')
flanks = self.bedtool.flank(
genome=self.assembly,
b=expansion_in_each_direction_Mb*1000000).sort().merge().saveas()
# compute the union of the flanks and the pathway
print('computing union')
union = self.bedtool.cat(flanks, postmerge=False).sort()
merged = union.merge().saveas()
print('total size after window addition:', merged.total_coverage(), 'bp')
self.bedtool = merged
def restricted_to_chrom_bedtool(self, chrnum):
return self.bedtool.filter(
lambda x : x[0] == 'chr' + str(int(chrnum))).saveas()
@classmethod
def reference_genome(cls, assembly='hg19'):
return GenomicSubset(assembly, path=paths.reference, assembly=assembly)
@classmethod
def reference_chrom_bedtool(cls, chrnum, assembly='hg19'):
return cls.reference_genome(assembly=assembly).restricted_to_chrom_bedtool(chrnum)
@classmethod
def whole_genome(cls, assembly='hg19'):
return cls(assembly, path=paths.reference)
def get_gene_dataframe(self, peak_bed, up=100000, down=100000):
# all overlap Enh-TSS(100000-tss-100000) pair with distance
peaks = BedTool(peak_bed)
b = BedTool(self.gene_bed)
b = b.flank(l=1, r=0, s=True, g=self.gsize).slop( # noqa: E741
l=up,
r=down,
g=self.gsize,
s=True # noqa: E741
)
# bedtools flank -r 0 -l 1 -i b.bed -g
# #all gene upstream 1bp position (TSS), Chr01 12800 12801 in Chr01 4170 12800 Xetrov90000001m.g 0 -
# bedtools slop -r down -l up -i b.bed -g
# # |100000--TSS--100000|
vals = []
# for f in b.intersect(peaks, wo=True, nonamecheck=True):
for f in b.intersect(peaks, wo=True):
# bedtools intersect -wo -nonamecheck -b peaks.bed -a b.bed
chrom = f[0]
strand = f[5]
if strand == "+":
tss = f.start + up
else:
tss = f.start + down
gene = f[3]
peak_start, peak_end = int(f[13]), int(f[14])
vals.append([chrom, tss, gene, peak_start, peak_end])
p = pd.DataFrame(
vals, columns=["chrom", "tss", "gene", "peak_start", "peak_end"])
p["peak"] = [int(i) for i in (p["peak_start"] + p["peak_end"]) / 2]
# peak with int function, let distance int
p["dist"] = np.abs(p["tss"] - p["peak"])
p["loc"] = (p["chrom"] + ":" + p["peak_start"].astype(str) + "-" +
p["peak_end"].astype(str))
p = p.sort_values("dist").drop_duplicates(
["loc", "gene"], keep="first")[["gene", "loc", "dist"]]
p = p[p["dist"] < up - 1]
# remove distance more than 100k interaction, for weight calculate
p.gene = [i.upper() for i in list(p.gene)]
return p
def get_promoter_dataframe(self, peak_bed, up=2000, down=2000):
# all overlap Enh-TSS(up2000 to down2000) pair
peaks = BedTool(peak_bed)
b = BedTool(self.gene_bed)
b = b.flank(l=1, r=0, s=True, g=self.gsize).slop( # noqa: E741
l=up,
r=down,
g=self.gsize,
s=True # noqa: E741
)
vals = []
# for f in b.intersect(peaks, wo=True, nonamecheck=True):
for f in b.intersect(peaks, wo=True):
chrom = f[0]
gene = f[3]
peak_start, peak_end = int(f[13]), int(f[14])
vals.append([chrom, gene, peak_start, peak_end])
prom = pd.DataFrame(
vals, columns=["chrom", "gene", "peak_start", "peak_end"])
prom["loc"] = (prom["chrom"] + ":" + prom["peak_start"].astype(str) +
"-" + prom["peak_end"].astype(str))
prom.gene = [i.upper() for i in list(prom.gene)]
return prom
def mk_matrix(inputfile=None,
outputfile=None,
bigwiglist=None,
ft_type=None,
pseudo_count=0,
upstream=1000,
downstream=1000,
bin_around_frac=0.1,
chrom_info=None,
bin_nb=100,
nb_proc=None,
labels=None,
no_stranded=False,
zero_to_na=False):
"""
Description: Create a matrix to be used by 'profile' and 'heatmap' commands.
"""
# -------------------------------------------------------------------------
# Check argument consistency
#
# -------------------------------------------------------------------------
if ft_type in ['single_nuc', 'promoter', 'tts']:
region_size = upstream + downstream + 1
if region_size < bin_nb:
message(
"The region (-u/-d) needs to be extended given the number "
"of bins (--bin-nb)",
type="ERROR")
# -------------------------------------------------------------------------
# Check output file name does not ends with .zip
#
# -------------------------------------------------------------------------
if outputfile.name.endswith(".zip"):
outfn = outputfile.name.replace(".zip", "")
outputfile = open(outfn, "w")
# -------------------------------------------------------------------------
# Check input file is in bed or GTF format
#
# -------------------------------------------------------------------------
message("Loading input file...")
if inputfile.name == '<stdin>':
gtf = GTF(inputfile.name)
is_gtf = True
if ft_type == 'user_regions':
message(
"--ft-type can not be set to user_regions"
" when a gtf is provided.",
type="ERROR")
else:
try:
region_bo = BedTool(inputfile.name)
len(region_bo)
except IndexError:
message("Unable to read the input file. Check format",
type="ERROR")
if len(region_bo) == 0:
message("Unable to find requested regions", type="ERROR")
if region_bo.file_type == 'gff':
message('Loading the GTF file.')
gtf = GTF(inputfile.name)
is_gtf = True
else:
is_gtf = False
if ft_type != 'user_regions' and ft_type != 'single_nuc':
message(
"Set --ft-type to 'user_regions' or 'single_nuc'"
" when using input bed file.",
type="ERROR")
# Check that the strand is provided and
# check it is located in the right column
# (not checked by BedTool...).
if region_bo.field_count() < 6:
if not no_stranded:
message("Strand is undefined. Use -nst.", type="ERROR")
else:
region_name = dict()
for i in region_bo:
if region_name.get(i.name, None) is None:
region_name[i.name] = 1
else:
message(
"Regions in bed file should have "
"unique identifier (col 4).",
type="ERROR")
if i.strand[0] not in ['.', '+', '-']:
message("Strand should be one of '+','-' or '.'.",
type="ERROR")
if ft_type == 'single_nuc':
if i.end - i.start != 1:
message(
"Region length should be 1 nucleotide "
"long when 'single_nuc' is set. Use 'user_regions'.",
type="ERROR")
elif ft_type == 'user_regions':
if i.end - i.start == 1:
message(
"Region length should not be 1 nucleotide "
"long when 'user_regions' is set. Use 'single_nuc'.",
type="ERROR")
# -------------------------------------------------------------------------
# Create a list of labels for the diagrams.
# Take user input in account
# -------------------------------------------------------------------------
message('Checking labels.')
if labels is not None:
labels = labels.split(",")
# Ensure the number of labels is the same as the number of bw files.
if len(labels) != len(bigwiglist):
message(
"The number of labels should be the same as the number of"
" bigwig files.",
type="ERROR")
# Ensure labels are non-redondant
if len(labels) > len(set(labels)):
message("Labels must be unique.", type="ERROR")
else:
labels = []
for i in range(len(bigwiglist)):
labels += [
os.path.splitext(os.path.basename(bigwiglist[i].name))[0]
]
# -------------------------------------------------------------------------
#
# Get the requested transcrit lines in bed format
# Tx are restricted to those found on chromosome
# declared in the bigwig file.
# -------------------------------------------------------------------------
message('Getting the list of chromosomes declared in bigwig files.')
bw_chrom = list()
for i in bigwiglist:
bw_chrom += list(pyBigWig.open(i.name).chroms().keys())
bed_col = [0, 1, 2, 3, 4, 5]
if is_gtf:
message('Selecting chromosomes declared in bigwig from gtf.')
tmp = gtf.select_by_key("feature", "transcript").select_by_key(
"seqid", ",".join(bw_chrom))
tmp = gtf.select_by_key("feature", "transcript")
tmp_tx_name = tmp.extract_data("transcript_id", as_list=True)
# If several trancript records are associated to
# the same transcript_id, raise an error.
if len(tmp_tx_name) > len(set(tmp_tx_name)):
message('Transcripts should have a unique identifier.',
type="ERROR")
message('Selecting requested regions.')
# ----------------------------------------------------------------------
#
# Slop tss and promoters.
# No need if transcript was requested (it will be flanked by upstream
# and doswnstream regions later on).
# ----------------------------------------------------------------------
if ft_type == 'transcript':
message("Getting transcript boundaries (input gtf).")
main_region_bo = tmp.to_bed(name=["transcript_id"])
elif ft_type == 'promoter':
message("Getting promoter regions [-%d,+%d]." %
(upstream, downstream))
main_region_bo = tmp.get_tss(name=["transcript_id"]).slop(
s=True, l=upstream, r=downstream, g=chrom_info.name)
elif ft_type == 'tts':
main_region_bo = tmp.get_tts(name=["transcript_id"]).slop(
s=True, l=upstream, r=downstream, g=chrom_info.name)
else:
message("Loading regions")
if ft_type == 'user_regions':
main_region_bo = BedTool(inputfile.name).cut(bed_col)
elif ft_type == 'single_nuc':
main_region_bo = BedTool(inputfile.name).cut(bed_col).slop(
s=True, l=upstream, r=downstream, g=chrom_info.name)
else:
message("Unknown method.")
# Save for tracability
main_region_bed = make_tmp_file(prefix="region" + ft_type, suffix=".bed")
main_region_bo.saveas(main_region_bed.name)
# -------------------------------------------------------------------------
#
# Print a header in the output file
#
# -------------------------------------------------------------------------
message("Preparing comments")
comments = "#"
comments += "ft_type:" + ft_type + ";"
comments += "from:" + str(upstream) + ";"
comments += "to:" + str(downstream) + ";"
comments += "labels:" + ",".join(labels) + ";"
# -------------------------------------------------------------------------
# Compute coverage of requested region
# Each worker will send a file
# -------------------------------------------------------------------------
outputfile_list = {}
message("Using %d bins for main region." % bin_nb)
tmp_file = bw_profile_mp(in_bed_file=main_region_bed.name,
nb_proc=nb_proc,
big_wig=[x.name for x in bigwiglist],
bin_nb=bin_nb,
pseudo_count=pseudo_count,
stranded=not no_stranded,
type="main",
labels=labels,
outputfile=outputfile.name,
zero_to_na=zero_to_na,
verbose=pygtftk.utils.VERBOSITY)
outputfile_list["main"] = tmp_file
# -------------------------------------------------------------------------
# If transcript was requested
# we must process flanking regions
# We need to retrieve coverage of promoter [-upstream, 0]
# as transcript coverage window size will depend on transcript length.
# For promoter the length of windows will be fixed.
# -------------------------------------------------------------------------
if ft_type in ['transcript', 'user_regions']:
# Number of bins for TTS and TSS
around_bin_nb = int(round(bin_nb * bin_around_frac))
if around_bin_nb < 1:
around_bin_nb = 1
if upstream > 0:
if ft_type == 'transcript':
message("Getting promoter (using %d bins)." % around_bin_nb)
ups_region_bo = tmp.get_tss(name=["transcript_id"]).slop(
s=True, l=upstream, r=-1, g=chrom_info.name).cut(bed_col)
else:
message("Getting upstream regions (%d bins)." % around_bin_nb)
ups_region_bo = main_region_bo.flank(s=True,
l=upstream,
r=0,
g=chrom_info.name)
upstream_bed_file = make_tmp_file(prefix="upstream_region" +
ft_type,
suffix=".bed")
ups_region_bo.saveas(upstream_bed_file.name)
tmp_file = bw_profile_mp(in_bed_file=upstream_bed_file.name,
nb_proc=nb_proc,
big_wig=[x.name for x in bigwiglist],
bin_nb=around_bin_nb,
pseudo_count=pseudo_count,
stranded=not no_stranded,
type="upstream",
labels=labels,
outputfile=outputfile.name,
zero_to_na=zero_to_na,
verbose=pygtftk.utils.VERBOSITY)
outputfile_list["upstream"] = tmp_file
if downstream > 0:
if ft_type == 'transcript':
message("Getting TTS (using %d bins)." % around_bin_nb)
dws_region_bo = tmp.get_tts(name=["transcript_id"]).slop(
s=True, l=-1, r=downstream, g=chrom_info.name).cut(bed_col)
else:
message("Getting downstream regions (%d bins)." %
around_bin_nb)
dws_region_bo = main_region_bo.flank(s=True,
l=0,
r=downstream,
g=chrom_info.name)
dws_bed_file = make_tmp_file(prefix="dowstream_region" + ft_type,
suffix=".bed")
dws_region_bo.saveas(dws_bed_file.name)
tmp_file = bw_profile_mp(in_bed_file=dws_bed_file.name,
nb_proc=nb_proc,
big_wig=[x.name for x in bigwiglist],
bin_nb=around_bin_nb,
pseudo_count=pseudo_count,
stranded=not no_stranded,
type="downstream",
labels=labels,
outputfile=outputfile.name,
zero_to_na=zero_to_na,
verbose=pygtftk.utils.VERBOSITY)
outputfile_list["downstream"] = tmp_file
# -------------------------------------------------------------------------
#
# Merge file using pandas
#
# -------------------------------------------------------------------------
message("Reading (pandas): " + outputfile_list["main"].name, type="DEBUG")
df_main = pd.read_csv(outputfile_list["main"].name, sep="\t")
# save strand and end
# They will re-joined added later
df_copy = df_main[['bwig', 'chrom', 'gene', 'strand', 'start', 'end']]
df_start = df_main.pop('start')
df_end = df_main.pop('end')
if "upstream" in outputfile_list:
message("Merging upstream file")
message("Reading (pandas): " + outputfile_list["upstream"].name,
type="DEBUG")
df_up = pd.read_csv(outputfile_list["upstream"].name, sep="\t")
df_up = df_up.drop(['start', 'end'], 1)
df_main = df_up.merge(df_main.loc[:, df_main.columns],
on=['bwig', 'chrom', 'gene', 'strand'])
if "downstream" in outputfile_list:
message("Merging downstream file")
message("Reading (pandas): " + outputfile_list["downstream"].name,
type="DEBUG")
df_dws = pd.read_csv(outputfile_list["downstream"].name, sep="\t")
df_dws = df_dws.drop(['start', 'end'], 1)
df_main = df_main.merge(df_dws.loc[:, df_dws.columns],
on=['bwig', 'chrom', 'gene', 'strand'])
# join start and end.
df_main = df_main.merge(df_copy.loc[:, df_copy.columns],
on=['bwig', 'chrom', 'gene', 'strand'])
df_start = df_main.pop('start')
df_end = df_main.pop('end')
df_main.insert(2, 'start', df_start)
df_main.insert(3, 'end', df_end)
message("Writing to file")
outputfile.close()
with open(outputfile.name, 'a') as f:
f.write(comments + "\n")
df_main.to_csv(f,
sep="\t",
index=False,
mode='a',
columns=df_main.columns,
na_rep='NA')
# -------------------------------------------------------------------------
#
# Compress
#
# -------------------------------------------------------------------------
message("Compressing")
path = os.path.abspath(outputfile.name)
filename = os.path.basename(path)
message("filename: " + filename, type="DEBUG")
zip_filename = filename + '.zip'
message("zip_filename: " + zip_filename, type="DEBUG")
zip_path = os.path.join(os.path.dirname(path), zip_filename)
message("zip_path: " + zip_path, type="DEBUG")
with zipfile.ZipFile(zip_path, 'w', allowZip64=True) as zf:
zf.write(filename=path, arcname=filename)
for i in outputfile_list:
message("deleting " + outputfile_list[i].name)
os.remove(outputfile_list[i].name)
os.remove(outputfile.name)
gc.disable()
close_properly(inputfile, outputfile)
temp_string = l_split[1] + '\t' + 'na' + '\t' + 'na' + '\t' + l_split[2] + '\t' + l_split[2] + '\t' + '.' + '\t' + '.' + '\t' + '.' + '\t' + l_split[0] + '\n'
gff_string = gff_string + temp_string
i += 1
target_gff = BedTool(gff_string, from_string=True)
## Annotation files.
print('Calculating annotations ...')
gencode_ann = BedTool(path_to_gencode_ann).sort()
protein_coding_genes_ann = gencode_ann.filter(lambda x: x[2] == 'gene').filter(lambda x: 'gene_type "protein_coding"' in x[8]).sort()
CGI_ann = BedTool(path_to_CGI).sort()
shore_ann = CGI_ann.flank(g=path_to_chr_lengths, b=2000).sort()
shelf_ann = CGI_ann.flank(g=path_to_chr_lengths, b=4000).subtract(shore_ann).sort()
ChrHMM_ann = BedTool(path_to_ChrHMM).sort()
## Intersections
print('Performing gene bodies / CGI intersections ...')
in_gene_bodies_cgs = list(set(list(target_gff.intersect(protein_coding_genes_ann).sort().to_dataframe()['attributes']))) # 15319 / 21368 CpGs are in gene bodies
in_CGI_cgs = list(set(list(target_gff.intersect(CGI_ann).sort().to_dataframe()['attributes']))) # 9319 / 21368 CpGs are in CGIs
in_shore_cgs = list(set(list(target_gff.intersect(shore_ann).sort().to_dataframe()['attributes']))) # 7920 / 21368 CpGs are in shores
in_shelf_cgs = list(set(list(target_gff.intersect(shelf_ann).sort().to_dataframe()['attributes']))) # 1138 / 21368 CpGs are in CGIs
# 2991 / 21368 CpGs are in open sea
def create(
cls: Type[T],
outdir: str,
data_files: List[str],
enhancer_file: str,
annotation_file: str,
genome: str,
window: Optional[int] = 2000,
anno_file: Optional[str] = None,
anno_from: Optional[str] = None,
anno_to: Optional[str] = None,
gene_mapping: Optional[str] = None,
threshold: Optional[float] = 1.0,
version: Optional[str] = "0.1.0",
) -> T:
outdir = Path(outdir)
basename = outdir.name
meanstd_file = outdir / f"{basename}.{genome}.meanstd.tsv.gz"
target_file = outdir / f"{basename}.{genome}.target.npz"
gene_file = outdir / "annotation.tss.merged1kb.bed"
link_file = outdir / "enhancers2genes.feather"
g = Genome(genome)
if not os.path.exists(outdir):
os.makedirs(outdir)
info = {
"genes": "genes.txt",
"enhancers": "enhancers.feather",
"link_file": os.path.basename(link_file),
"genome": genome,
"window": window,
"meanstd_file": os.path.basename(meanstd_file),
"target_file": os.path.basename(target_file),
"gene_file": os.path.basename(gene_file),
"version": version,
"schema_version": __schema_version__,
}
if anno_file is not None:
if not os.path.exists(anno_file):
raise ValueError(f"{anno_file} does not exist")
if anno_from is None or anno_to is None:
raise ValueError("Need anno_from and anno_to columns!")
copyfile(anno_file, outdir / os.path.basename(anno_file))
info.update({
"anno_file": os.path.basename(anno_file),
"anno_from": anno_from,
"anno_to": anno_to,
})
if gene_mapping is not None:
if not os.path.exists(gene_mapping):
raise ValueError(f"{gene_mapping} does not exist")
copyfile(gene_mapping, outdir / os.path.basename(gene_mapping))
info["gene_mapping"] = os.path.basename(gene_mapping)
logger.info("processing gene annotation")
# Convert gene annotation
b = BedTool(annotation_file)
chroms = set([f.chrom for f in BedTool(enhancer_file)])
b = b.filter(lambda x: x.chrom in chroms)
b = (b.flank(g=g.sizes_file, l=1,
r=0).sort().merge(d=1000, c=4,
o="distinct")) # noqa: E741
b.saveas(str(gene_file))
logger.info("processing data files")
# create coverage_table
df = coverage_table(
enhancer_file,
data_files,
window=window,
log_transform=True,
normalization="quantile",
ncpus=12,
)
df.index.rename("loc", inplace=True)
df.reset_index().to_feather(f"{outdir}/enhancers.feather")
np.savez(target_file, target=df.iloc[:, 0].sort_values())
meanstd = pd.DataFrame(index=df.index, )
meanstd["mean"] = df.mean(1)
meanstd["std"] = df.std(1)
meanstd = meanstd.reset_index().rename(columns={"loc": "index"})
meanstd.to_csv(meanstd_file, compression="gzip", index=False, sep="\t")
df.index.rename("loc", inplace=True)
df = df.sub(df.mean(1), axis=0)
df = df.div(df.std(1), axis=0)
df.reset_index().to_feather(f"{outdir}/enhancers.feather")
link = create_link_file(meanstd_file, gene_file, genome=genome)
link.to_feather(link_file)
genes = _create_gene_table(
df,
meanstd_file,
gene_file,
gene_mapping,
genome=genome,
link_file=link_file,
threshold=threshold,
)
genes.to_csv(f"{outdir}/genes.txt", sep="\t")
with open(f"{outdir}/info.yaml", "w") as f:
yaml.dump(info, f)
return ScepiaDataset(outdir)
import numpy as np
from scipy import stats
from pybedtools import BedTool
def filter_chipseq_files(infile):
df = pd.read_table(infile,
sep='\t',
header=None,
names=[
'chrom', 'start', 'end', 'name', 'score', 'strand',
'signal', 'p', 'q', 'peak'
])
df['signal_zscore'] = stats.zscore(df.signal)
fltr_df = df[df.signal_zscore > 1.64]
return BedTool.from_dataframe(fltr_df)
# make promoter file | general promoter set
genes = BedTool('rnaseq_ensembl_genemodels_filter.bed')
tss_window = genes.flank(l=2000, r=0, s=True, genome='hg19')
tss_window.saveas('hg19_tss_2kb.bed')
# make promoter file | requires overlap with high signal H3K27ac/H3K4me3
h3k27ac = filter_chipseq_files('E073-H3K27ac.narrowPeak.gz')
h3k4me3 = filter_chipseq_files('E073-H3K4me3.narrowPeak.gz')
tss_high_conf = (tss_window.intersect(h3k27ac, u=True)).intersect(h3k4me3,
u=True)
tss_high_conf.saveas('hg19_tss_high_conf.bed')
