def make_file_for_primer_3(gff_file, ref_file, names_file, output_file, start,
end):
# check for a tmp direcory
if len(glob.glob("./tmp")) == 0:
call(["mkdir", "tmp"])
gff_file = list(annotation.read_annotations(gff_file))
print "\nReading in the reference file\n"
seq_dict = dict(io.read_sequences(ref_file))
names_file = open(names_file).readlines()
config = open("Software/primer_config.txt").readlines()
with open("tmp/regions_" + output_file, 'w') as out_f:
for name in names_file:
sname = name.strip("\n ")
found = False
for line in gff_file:
gff_name = line.attr.get("Name", "No_name")
peak = line.attr.get("id", "No_id")
if sname in gff_name.split("/"):
out_f.write("SEQUENCE_ID=" + gff_name.replace("/", "_") +
"_" + peak + "\n")
# Move the peaks 30 bases proximal
out_f.write("SEQUENCE_TEMPLATE=" +
line.shifted(start, end).get_seq(seq_dict) +
"\n")
found = True
for cline in config:
out_f.write(cline.strip("\n") + "\n")
out_f.write("=" + "\n")
if found == False:
print "Could not find the gene " + sname + " in the reference gff file\n"
def run(self):
if self.output is not None:
out_file = open(self.output,'wb')
else:
out_file = sys.stdout
annotation.write_gff3_header(out_file)
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if 'ID' not in item.attr and 'locus_tag' in item.attr:
item.attr['ID'] = item.attr['locus_tag']
if 'color' not in item.attr:
if item.type == 'CDS':
item.attr['color'] = '#008800'
if item.type == 'rRNA':
item.attr['color'] = '#bb0000'
if item.type == 'tRNA':
item.attr['color'] = '#bb00bb'
if item.type == 'misc_feature':
item.attr['color'] = '#8888ff'
print >> out_file, item.as_gff()
if self.output is not None:
out_file.close()
def set_annotations(self, filenames):
f = self.open('reference.gff','wb')
annotation.write_gff3_header(f)
for filename in filenames:
for feature in annotation.read_annotations(filename):
print >> f, feature.as_gff()
f.close()
def set_annotations(self, filenames):
f = self.open('reference.gff','wb')
print >> f, '##gff-version 3'
for filename in filenames:
for feature in annotation.read_annotations(filename):
print >> f, feature.as_gff()
f.close()
def build_snpeff(self):
jar = io.find_jar('snpEff.jar')
with open(self/'snpeff.config','wb') as f:
print >> f, 'data_dir = snpeff'
print >> f, 'genomes : ' + self.name
print >> f, self.name + '.genome : ' + self.name
snpwork = io.Workspace(self/'snpeff',must_exist=False)
snpwork_genome = io.Workspace(snpwork/self.name,must_exist=False)
snpwork_genomes = io.Workspace(snpwork/'genomes',must_exist=False)
annotations = self.annotations_filename()
assert annotations
with open(snpwork_genome/'genes.gff','wb') as f:
for record in annotation.read_annotations(annotations):
if record.end <= record.start: continue
if not record.attr:
record.attr['attributes'] = 'none'
print >> f, record.as_gff()
with open(snpwork_genomes/(self.name+'.fa'),'wb') as f:
for name, seq in io.read_sequences(self.reference_fasta_filename()):
io.write_fasta(f, name, seq)
io.execute('java -jar JAR build NAME -gff3 -c CONFIG',
JAR=jar, NAME=self.name, CONFIG=self/'snpeff.config')
def coding_regions(self):
coding_regions = {}
annotations = list(
annotation.read_annotations(join(self.dirname, 'reference.gff')))
annotation.link_up_annotations(annotations)
for item in annotations:
if item.type == "CDS":
[mrna] = item.parents
[gene] = mrna.parents
name = gene.get_id()
if name not in coding_regions:
coding_regions[name] = []
coding_regions[name].append(item)
for gene in annotations:
if gene.type != "gene": continue
name = gene.get_id()
if name not in coding_regions:
coding_region = gene.three_prime()
coding_region.attr = {"ID": name}
else:
items = coding_regions[name]
coding_region = annotation.Annotation(
type="coding_region",
seqid=items[0].seqid,
strand=items[0].strand,
start=min(item2.start for item2 in items),
end=max(item2.end for item2 in items),
attr={"ID": name},
)
coding_regions[name] = coding_region
return coding_regions
def set_annotations(self, filenames):
f = self.open('reference.gff', 'wb')
print >> f, '##gff-version 3'
for filename in filenames:
for feature in annotation.read_annotations(filename):
print >> f, feature.as_gff()
f.close()
def run(self):
only = set( item.lower() for item in self.only )
exclude = set( item.lower() for item in self.exclude )
if self.output is not None:
out_file = open(self.output,'wb')
else:
out_file = sys.stdout
print >> out_file, '##gff-version 3'
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if only and item.type.lower() not in only: continue
if item.type.lower() in exclude: continue
if 'ID' not in item.attr and 'locus_tag' in item.attr:
item.attr['ID'] = item.attr['locus_tag']
if 'color' not in item.attr:
if item.type == 'CDS':
item.attr['color'] = '#008800'
if item.type == 'rRNA':
item.attr['color'] = '#bb0000'
if item.type == 'tRNA':
item.attr['color'] = '#bb00bb'
if item.type == 'misc_feature':
item.attr['color'] = '#8888ff'
print >> out_file, item.as_gff()
if self.output is not None:
out_file.close()
def run(self):
extractions = [ ]
for item in self.genes.split(','):
extraction = item.split('/')
assert len(extraction) == 4
extractions.append(extraction)
rename = { }
if self.rename:
for item in self.rename.split(','):
old,new = item.split('=')
rename[old] = new
work = self.get_workspace()
with workspace.tempspace() as temp:
items = list(annotation.read_annotations(self.annotation))
for item in items:
item.seqid = rename.get(item.seqid, item.seqid)
annotation.write_gff3(temp/'temp.gff', get_genes(items, extractions, self.log))
del items
with open(temp/'temp.fa','wb') as f:
for name,seq in io.read_sequences(self.genome):
name = name.split()[0]
name = rename.get(name,name)
io.write_fasta(f, name, seq)
reference_directory.Make_tt_reference(
self.output_dir,
filenames = [ temp/'temp.fa', temp/'temp.gff' ],
index = self.index,
).run()
def _describe_peaks(self, r):
workspace = io.Workspace(self.output_dir, must_exist=False)
counts = io.read_grouped_table(workspace/("expression","peakwise","counts.csv"))["Count"]
peak_counts = collections.defaultdict(int)
read_counts = collections.defaultdict(int)
total = 0
for item in annotation.read_annotations(workspace/("peaks","relation-child.gff")):
peak_counts[item.attr.get("Relation","None")] += 1
read_counts[item.attr.get("Relation","None")] += sum(int(c) for c in counts[item.get_id()].values())
total += 1
total_reads = sum(read_counts.values())
r.write("<p>\n")
r.write("%d peaks\n" % total)
for name, desc in [
("3'UTR", "in a 3' UTR"),
("Exon", "otherwise in an exon"),
("Downstrand", "otherwise downstrand of a non-coding RNA"),
("Intron", "otherwise in an intron"),
("Antisense", "otherwise antisense to a gene"),
("None", "couldn't be related to annotated genes"),
]:
r.write("<br/>%d peaks and %.1f%% of reads %s\n" % (peak_counts[name], read_counts[name]*100.0/total_reads, desc))
r.write("</p>\n")
def make_file_for_primer_3 (gff_file, ref_file, names_file, output_file):
# check for a tmp direcory
if len(glob.glob("./tmp")) == 0:
call (["mkdir", "tmp"])
gff_file = list(annotation.read_annotations(gff_file))
print "\n Reading in the reference file"
seq_dict = dict(io.read_sequences(ref_file))
names_file = open(names_file).readlines()
config = open("primer_config.txt").readlines()
with open("tmp/regions_" + output_file, 'w') as out_f:
for name in names_file:
sname = name.strip("\n")
found = False
for line in gff_file:
gff_name = line.attr.get ("Name", "No_name")
peak = line.attr.get ("id", "No_id")
if sname in gff_name.split("/"):
out_f.write ("SEQUENCE_ID="+ gff_name.replace("/", "_") + "_" + peak + "\n")
out_f.write("SEQUENCE_TEMPLATE=" + line.shifted(-100, 0).get_seq(seq_dict) + "\n")
found = True
for cline in config:
out_f.write(cline.strip("\n") + "\n")
out_f.write("=" + "\n")
if found ==False:
print "Could not find the gene " + sname + " in the gff file"
def run(self):
if self.output is not None:
out_file = open(self.output, 'wb')
else:
out_file = sys.stdout
annotation.write_gff3_header(out_file)
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if 'ID' not in item.attr and 'locus_tag' in item.attr:
item.attr['ID'] = item.attr['locus_tag']
if 'color' not in item.attr:
if item.type == 'CDS':
item.attr['color'] = '#008800'
if item.type == 'rRNA':
item.attr['color'] = '#bb0000'
if item.type == 'tRNA':
item.attr['color'] = '#bb00bb'
if item.type == 'misc_feature':
item.attr['color'] = '#8888ff'
print >> out_file, item.as_gff()
if self.output is not None:
out_file.close()
def run(self):
extractions = [ ]
for item in self.genes.split(','):
extraction = item.split('/')
assert len(extraction) == 4
extractions.append(extraction)
rename = { }
if self.rename:
for item in self.rename.split(','):
old,new = item.split('=')
rename[old] = new
work = self.get_workspace()
with workspace.tempspace() as temp:
items = list(annotation.read_annotations(self.annotation))
for item in items:
item.seqid = rename.get(item.seqid, item.seqid)
annotation.write_gff3(temp/'temp.gff', get_genes(items, extractions, self.log))
del items
with open(temp/'temp.fa','wb') as f:
for name,seq in io.read_sequences(self.genome):
name = name.split()[0]
name = rename.get(name,name)
io.write_fasta(f, name, seq)
reference_directory.Make_tt_reference(
self.output_dir,
filenames = [ temp/'temp.fa', temp/'temp.gff' ] + self.extra,
index = self.index, shrimp = self.shrimp,
bowtie = self.bowtie, star = self.star
).run()
def _describe_peaks(self, r):
workspace = io.Workspace(self.output_dir, must_exist=False)
counts = io.read_grouped_table(workspace/("expression","peakwise","counts.csv"))["Count"]
peak_counts = collections.defaultdict(int)
read_counts = collections.defaultdict(int)
total = 0
for item in annotation.read_annotations(workspace/("peaks","relation-child.gff")):
peak_counts[item.attr.get("Relation","None")] += 1
read_counts[item.attr.get("Relation","None")] += sum(int(c) for c in counts[item.get_id()].values())
total += 1
total_reads = sum(read_counts.values())
r.write("<p>\n")
r.write("%d peaks\n" % total)
for name, desc in [
("3'UTR", "in a 3' UTR"),
("Exon", "otherwise in an exon"),
("Downstrand", "otherwise downstrand of a non-coding RNA"),
("Intron", "otherwise in an intron"),
("Antisense", "otherwise antisense to a gene"),
("None", "couldn't be related to annotated genes"),
]:
r.write("<br/>%d peaks and %.1f%% of reads %s\n" % (peak_counts[name], read_counts[name]*100.0/total_reads, desc))
r.write("</p>\n")
def run(self):
only = set(item.lower() for item in self.only)
exclude = set(item.lower() for item in self.exclude)
if self.output is not None:
out_file = open(self.output, 'wb')
else:
out_file = sys.stdout
print >> out_file, '##gff-version 3'
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if only and item.type.lower() not in only: continue
if item.type.lower() in exclude: continue
if 'ID' not in item.attr and 'locus_tag' in item.attr:
item.attr['ID'] = item.attr['locus_tag']
if 'color' not in item.attr:
if item.type == 'CDS':
item.attr['color'] = '#008800'
if item.type == 'rRNA':
item.attr['color'] = '#bb0000'
if item.type == 'tRNA':
item.attr['color'] = '#bb00bb'
if item.type == 'misc_feature':
item.attr['color'] = '#8888ff'
print >> out_file, item.as_gff()
if self.output is not None:
out_file.close()
def coding_regions(self):
coding_regions = { }
annotations = list(annotation.read_annotations(join(self.dirname,'reference.gff')))
annotation.link_up_annotations(annotations)
for item in annotations:
if item.type == "CDS":
[ mrna ] = item.parents
[ gene ] = mrna.parents
name = gene.get_id()
if name not in coding_regions:
coding_regions[name] = [ ]
coding_regions[name].append(item)
for gene in annotations:
if gene.type != "gene": continue
name = gene.get_id()
if name not in coding_regions:
coding_region = gene.three_prime()
coding_region.attr = { "ID" : name }
else:
items = coding_regions[name]
coding_region = annotation.Annotation(
type = "coding_region",
seqid = items[0].seqid,
strand= items[0].strand,
start = min(item2.start for item2 in items),
end = max(item2.end for item2 in items),
attr = { "ID" : name },
)
coding_regions[name] = coding_region
return coding_regions
def run(self):
features_parent = [
_Related_feature(item, item.start, item.end, [])
for item in annotation.read_annotations(self.parent)
if selection.matches(self.select_parent, [item.type])
]
features_child = [
_Related_feature(item, item.start, item.end, [])
for item in annotation.read_annotations(self.child)
if selection.matches(self.select_child, [item.type])
]
index = {}
for item in features_child:
if item.feature.seqid not in index:
index[item.feature.seqid] = span_index.Span_index()
index[item.feature.seqid].insert(item)
for value in index.values():
value.prepare()
for item_1 in features_parent:
if item_1.feature.strand == 1:
start = item_1.start - self.upstrand
end = item_1.end + self.downstrand
elif item_1.feature.strand == -1:
start = item_1.start - self.downstrand
end = item_1.end + self.upstrand
else:
start = item_1.start - max(self.upstrand, self.downstrand)
end = item_1.end + max(self.upstrand, self.downstrand)
if item_1.feature.seqid in index:
for item_2 in index[item_1.feature.seqid].get(start, end):
item_1.relations.append(item_2)
item_2.relations.append(item_1)
for item in features_parent:
item.modify_with_relations(self.use, self.to_child, self.to_parent)
with open(self.prefix + '-parent.gff', 'wb') as f:
annotation.write_gff3_header(f)
for item in features_parent:
print >> f, item.feature.as_gff()
with open(self.prefix + '-child.gff', 'wb') as f:
annotation.write_gff3_header(f)
for item in features_child:
print >> f, item.feature.as_gff()
def run(self):
features_parent = [
_Related_feature(item,item.start,item.end,[])
for item in annotation.read_annotations(self.parent)
if selection.matches(self.select_parent, [item.type])
]
features_child = [
_Related_feature(item,item.start,item.end,[])
for item in annotation.read_annotations(self.child)
if selection.matches(self.select_child, [item.type])
]
index = { }
for item in features_child:
if item.feature.seqid not in index:
index[item.feature.seqid] = span_index.Span_index()
index[item.feature.seqid].insert(item)
for value in index.values():
value.prepare()
for item_1 in features_parent:
if item_1.feature.strand == 1:
start = item_1.start - self.upstrand
end = item_1.end + self.downstrand
elif item_1.feature.strand == -1:
start = item_1.start - self.downstrand
end = item_1.end + self.upstrand
else:
start = item_1.start - max(self.upstrand,self.downstrand)
end = item_1.end + max(self.upstrand,self.downstrand)
if item_1.feature.seqid in index:
for item_2 in index[item_1.feature.seqid].get(start,end):
item_1.relations.append(item_2)
item_2.relations.append(item_1)
for item in features_parent:
item.modify_with_relations(self.use, self.to_child, self.to_parent)
with open(self.prefix + '-parent.gff','wb') as f:
annotation.write_gff3_header(f)
for item in features_parent:
print >> f, item.feature.as_gff()
with open(self.prefix + '-child.gff','wb') as f:
annotation.write_gff3_header(f)
for item in features_child:
print >> f, item.feature.as_gff()
def index(filename, type=None, modify = lambda item: item, name = lambda item: item.get_id()):
result = { }
for item in annotation.read_annotations(filename):
if type is not None and item.type != type: continue
item = modify(item)
assert name(item) not in result
result[name(item)] = item
return result
def index(filename,
type=None,
modify=lambda item: item,
name=lambda item: item.get_id()):
result = collections.OrderedDict()
for item in annotation.read_annotations(filename):
if type is not None and item.type != type: continue
item = modify(item)
assert name(item) not in result
result[name(item)] = item
return result
def run(self):
assert self.change_strand in STRAND_CHANGE, 'Unknown way to change strand.'
strand_changer = STRAND_CHANGE[self.change_strand]
shift_start_absolute, shift_start_proportion = decode_shift(
self.shift_start)
shift_end_absolute, shift_end_proportion = decode_shift(self.shift_end)
renames = []
if self.rename:
for item in self.rename.split(','):
new, old = item.split('=')
if new != old:
renames.append((new, old))
out_file = open(self.prefix + '.gff', 'wb')
annotation.write_gff3_header(out_file)
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if self.type:
item.type = self.type
length = item.end - item.start
shift_start = int(
math.floor(0.5 + shift_start_absolute +
shift_start_proportion * length))
shift_end = int(
math.floor(0.5 + shift_end_absolute +
shift_end_proportion * length))
if item.strand == 1:
item.start += shift_start
item.end += shift_end
elif item.strand == -1:
item.end -= shift_start
item.start -= shift_end
item.start = max(0, item.start) #IGV complains
item.strand = strand_changer[item.strand]
old_attr = item.attr.copy()
for new, old in renames:
if old in item.attr:
del item.attr[old]
for new, old in renames:
if old in old_attr:
item.attr[new] = old_attr[old]
print >> out_file, item.as_gff()
out_file.close()
def run(self):
annotations = [ ]
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if self.type:
item.type = self.type
annotations.append(item)
annotations.sort(key=lambda item: (item.seqid, item.strand, item.start))
group = [ ]
groups = [ ]
def emit():
if not group: return
groups.append(group[:])
del group[:]
seqid = None
strand = None
end = 0
for item in annotations:
if item.seqid != seqid or item.strand != strand or item.start >= end:
emit()
seqid = item.seqid
strand = item.strand
end = item.end-self.overlap
group.append(item)
end = max(item.end-self.overlap, end)
emit()
out_file = open(self.prefix+'.gff','wb')
annotation.write_gff3_header(out_file)
for group in groups:
item = annotation.Annotation()
item.source = group[0].source
item.type = join_descriptions( item2.type for item2 in group )
item.seqid = group[0].seqid
item.strand = group[0].strand
item.start = min( item2.start for item2 in group )
item.end = max( item2.end for item2 in group )
item.score = None
item.phase = None
item.attr = { }
for item2 in group:
for key in item2.attr:
if key in item.attr: continue
item.attr[key] = join_descriptions( item3.attr[key] for item3 in group if key in item3.attr )
print >> out_file, item.as_gff()
out_file.close()
def run(self):
assert self.release
assert self.species
assert self.assembly
assert self.dna
extractions = [ ]
for item in self.genes.split(','):
extraction = item.split('/')
assert len(extraction) == 4
extractions.append(extraction)
rename = { }
if self.rename:
for item in self.rename.split(','):
old,new = item.split('=')
rename[old] = new
work = self.get_workspace()
ensembl = workspace.Workspace(work/'ensembl')
genome_filename = self.species+"."+self.assembly+"."+self.dna+".fa.gz"
genome_url = "rsync://ftp.ensembl.org/ensembl/pub/release-"+self.release+"/fasta/"+self.species.lower()+"/dna/"+genome_filename
gff_filename = self.species+"."+self.assembly+"."+self.release+".gff3.gz"
gff_url = "rsync://ftp.ensembl.org/ensembl/pub/release-"+self.release+"/gff3/"+self.species.lower()+"/"+gff_filename
if self.download:
self.log.log("Fetching "+genome_url+"\n")
io.execute(['rsync','-aP',genome_url, ensembl/genome_filename])
self.log.log("Fetching "+gff_url+"\n")
io.execute(['rsync','-aP',gff_url, ensembl/gff_filename])
with workspace.tempspace() as temp:
items = list(annotation.read_annotations(ensembl/gff_filename))
for item in items:
item.seqid = rename.get(item.seqid, item.seqid)
annotation.write_gff3(temp/'temp.gff', get_genes(items, extractions, self.log))
del items
with open(temp/'temp.fa','wb') as f:
for name,seq in io.read_sequences(ensembl/genome_filename):
name = name.split()[0]
name = rename.get(name,name)
io.write_fasta(f, name, seq)
reference_directory.Make_tt_reference(
self.output_dir,
filenames = [ temp/'temp.fa', temp/'temp.gff' ],
index = self.index,
).run()
def run(self):
f = self.begin_output()
for filename in self.filenames:
info = io.get_file_info(filename)
any = False
name = os.path.splitext(os.path.split(filename)[1])[0]
if info.matches('sequences'):
total = 0
total_length = 0
for seq in io.read_sequences(filename, qualities=True):
total += 1
total_length += len(seq[1])
print >> f, grace.datum(name, 'sequences', total)
print >> f, grace.datum(name, 'total bases', total_length)
if total:
print >> f, grace.datum(name, 'average length',
float(total_length) / total)
print >> f
any = True
if info.matches('annotations'):
total = 0
counts = {}
for item in annotation.read_annotations(filename, "/"):
total += 1
counts[item.type] = counts.get(item.type, 0) + 1
print >> f, grace.datum(name, 'features', total)
for key in sorted(counts):
print >> f, grace.datum(name, key + ' features',
counts[key])
print >> f
any = True
if info.matches('type-vcf'):
reader_f = io.open_possibly_compressed_file(filename)
reader = vcf.Reader(reader_f)
n = 0
for item in reader:
n += 1
print >> f, grace.datum(name, 'variants', n)
any = True
if not any:
raise grace.Error('Don\'t know what to do with ' + filename)
self.end_output(f)
def run(self):
f = self.begin_output()
for filename in self.filenames:
any = False
name = os.path.splitext(os.path.split(filename)[1])[0]
try:
iterator = io.read_sequences(filename, qualities=True)
except grace.Error:
iterator = None
if iterator is not None:
total = 0
total_length = 0
for seq in io.read_sequences(filename, qualities=True):
total += 1
total_length += len(seq[1])
print >> f, grace.datum(name, 'sequences', total)
print >> f, grace.datum(name, 'average length',
float(total_length) / total)
print >> f
any = True
try:
iterator = annotation.read_annotations(filename)
except grace.Error:
iterator = None
if iterator:
total = 0
counts = {}
for item in iterator:
total += 1
counts[item.type] = counts.get(item.type, 0) + 1
print >> f, grace.datum(name, 'features', total)
for key in sorted(counts):
print >> f, grace.datum(name, key + ' features',
counts[key])
print >> f
any = True
if not any:
raise grace.Error(
filename +
' is neither a sequence file nor an annotation file that nesoni can read.'
)
self.end_output(f)
def run(self):
assert self.change_strand in STRAND_CHANGE, 'Unknown way to change strand.'
strand_changer = STRAND_CHANGE[self.change_strand]
shift_start_absolute, shift_start_proportion = decode_shift(self.shift_start)
shift_end_absolute, shift_end_proportion = decode_shift(self.shift_end)
renames = [ ]
if self.rename:
for item in self.rename.split(','):
new, old = item.split('=')
if new != old:
renames.append((new,old))
out_file = open(self.prefix+'.gff','wb')
annotation.write_gff3_header(out_file)
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if self.type:
item.type = self.type
length = item.end-item.start
shift_start = int(math.floor(0.5+shift_start_absolute+shift_start_proportion*length))
shift_end = int(math.floor(0.5+shift_end_absolute+shift_end_proportion*length))
if item.strand == 1:
item.start += shift_start
item.end += shift_end
elif item.strand == -1:
item.end -= shift_start
item.start -= shift_end
item.start = max(0, item.start) #IGV complains
item.strand = strand_changer[item.strand]
old_attr = item.attr.copy()
for new,old in renames:
if old in item.attr:
del item.attr[old]
for new,old in renames:
if old in old_attr:
item.attr[new] = old_attr[old]
print >> out_file, item.as_gff()
out_file.close()
def run(self):
f = self.begin_output()
for filename in self.filenames:
info = io.get_file_info(filename)
any = False
name = os.path.splitext(os.path.split(filename)[1])[0]
if info.matches('sequences'):
total = 0
total_length = 0
for seq in io.read_sequences(filename, qualities=True):
total += 1
total_length += len(seq[1])
print >> f, grace.datum(name, 'sequences', total)
print >> f, grace.datum(name, 'total bases', total_length)
if total:
print >> f, grace.datum(name, 'average length', float(total_length)/total)
print >> f
any = True
if info.matches('annotations'):
total = 0
counts = { }
for item in annotation.read_annotations(filename):
total += 1
counts[item.type] = counts.get(item.type,0)+1
print >> f, grace.datum(name, 'features', total)
for key in sorted(counts):
print >> f, grace.datum(name, key + ' features', counts[key])
print >> f
any = True
if info.matches('type-vcf'):
reader_f = io.open_possibly_compressed_file(filename)
reader = vcf.Reader(reader_f)
n = 0
for item in reader:
n += 1
print >> f, grace.datum(name, 'variants', n)
any = True
if not any:
raise grace.Error('Don\'t know what to do with ' + filename)
self.end_output(f)
def run(self):
f = self.begin_output()
for filename in self.filenames:
any = False
name = os.path.splitext(os.path.split(filename)[1])[0]
try:
iterator = io.read_sequences(filename, qualities=True)
except grace.Error:
iterator = None
if iterator is not None:
total = 0
total_length = 0
for seq in io.read_sequences(filename, qualities=True):
total += 1
total_length += len(seq[1])
print >> f, grace.datum(name, 'sequences', total)
print >> f, grace.datum(name, 'average length', float(total_length)/total)
print >> f
any = True
try:
iterator = annotation.read_annotations(filename)
except grace.Error:
iterator = None
if iterator:
total = 0
counts = { }
for item in iterator:
total += 1
counts[item.type] = counts.get(item.type,0)+1
print >> f, grace.datum(name, 'features', total)
for key in sorted(counts):
print >> f, grace.datum(name, key + ' features', counts[key])
print >> f
any = True
if not any:
raise grace.Error(filename + ' is neither a sequence file nor an annotation file that nesoni can read.')
self.end_output(f)
def run(self):
assert self.change_strand in STRAND_CHANGE, 'Unknown way to change strand.'
strand_changer = STRAND_CHANGE[self.change_strand]
out_file = open(self.prefix+'.gff','wb')
annotation.write_gff3_header(out_file)
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if item.strand == 1:
item.start += self.shift_start
item.end += self.shift_end
elif item.strand == -1:
item.end -= self.shift_start
item.start -= self.shift_end
item.strand = strand_changer[item.strand]
print >> out_file, item.as_gff()
out_file.close()
def run(self):
work = self.get_workspace()
work.update_param(remove=['tail_tools_reference_version'])
nesoni.Make_reference(
self.output_dir,
filenames = self.filenames,
snpeff = False,
cs = 'ifavailable' if self.index else False,
ls = False,
bowtie = 'ifavailable' if self.index else False,
).run()
annotations = list(annotation.read_annotations(work/'reference.gff'))
annotation.link_up_annotations(annotations)
exon_index = span_index.index_annotations([
item for item in annotations if item.type == "exon"
])
mrna_end_index = span_index.index_annotations([
item.three_prime() for item in annotations if item.type == "mRNA"
])
mrna_utrs = [ ]
gene_utrs = [ ]
for gene in annotations:
if gene.type != 'gene': continue
mrnas = [ item for item in gene.children if item.type == 'mRNA' ]
assert mrnas, "Gene without any mRNAs: "+gene.get_id()
gene.attr['color'] = '#880088'
gene.start = min(item.start for item in mrnas)
gene.end = max(item.end for item in mrnas)
gene.attr["max_extension"] = str(_max_extension(gene, exon_index, mrna_end_index))
gene_utr_5primes = [ ]
for mrna in mrnas:
assert mrna.strand == gene.strand, mrna
assert mrna.seqid == gene.seqid, mrna
mrna.attr["max_extension"] = str(_max_extension(mrna, exon_index, mrna_end_index))
cdss = [ item for item in mrna.children if item.type == 'CDS' ]
exons = [ item for item in mrna.children if item.type == 'exon' ]
if not exons: continue
#link up annotations sorts children, so final is really final
for item in exons[:-1]:
item.attr["max_extension"] = "0"
exons[-1].attr["max_extension"] = mrna.attr["max_extension"]
if not cdss: continue
mrna_utr_5primes = [ ]
if gene.strand >= 0:
cds_3prime = max(item.end for item in cdss)
for item in exons:
if item.end >= cds_3prime:
mrna_utr_5primes.append(max(item.start,cds_3prime))
else:
cds_3prime = min(item.start for item in cdss)
for item in exons:
if item.start <= cds_3prime:
mrna_utr_5primes.append(min(item.end,cds_3prime))
if mrna.strand >= 0:
utr_start = min(mrna_utr_5primes) if mrna_utr_5primes else mrna.end
utr_end = max(utr_start+1,mrna.end)
gene_utr_5primes.append(utr_start)
else:
utr_end = max(mrna_utr_5primes) if mrna_utr_5primes else mrna.start
utr_start = min(mrna.start,utr_end-1)
gene_utr_5primes.append(utr_end)
attr = mrna.attr.copy()
attr['Parent'] = attr['ID']
attr['ID'] = attr['ID']+'-3UTR'
attr['color'] = '#008888'
utr = annotation.Annotation(
source = 'tt',
type = 'three_prime_utr',
seqid = mrna.seqid,
strand = mrna.strand,
start = utr_start,
end = utr_end,
attr = attr,
)
max_ext = _max_extension(utr, exon_index, mrna_end_index)
utr.attr["max_extension"] = str(max_ext)
#Only include if there is an annotated 3' UTR or end is not in the middle of some other isoform's exon
if utr_end-utr_start+max_ext > 1:
mrna_utrs.append(utr)
if gene.strand >= 0:
utr_start = max(gene_utr_5primes) if gene_utr_5primes else gene.end
utr_end = max(utr_start+1,gene.end)
else:
utr_end = min(gene_utr_5primes) if gene_utr_5primes else gene.start
utr_start = min(gene.start,utr_end-1)
attr = gene.attr.copy()
attr['Parent'] = attr['ID']
attr['ID'] = attr['ID']+'-3UTR'
attr['color'] = '#008888'
utr = annotation.Annotation(
source = 'tt',
type = 'three_prime_utr',
seqid = gene.seqid,
strand = gene.strand,
start = utr_start,
end = utr_end,
attr = attr,
)
utr.attr["max_extension"] = str(_max_extension(utr, exon_index, mrna_end_index))
gene_utrs.append(utr)
annotation.write_gff3(work/'reference.gff', annotations + mrna_utrs)
annotation.write_gff3(work/'utr.gff', gene_utrs)
work.update_param(tail_tools_reference_version=work.VERSION)
def run(self):
assert self.extension is not None, '--extension must be specified'
#workspace = self.get_workspace()
workspace = working_directory.Working(self.working_dir,
must_exist=True)
if self.annotations == None:
reference = workspace.get_reference()
annotations_filename = reference.annotations_filename()
else:
annotations_filename = self.annotations
types = [item.lower() for item in self.types.split(',')]
parts = self.parts or self.types
parts = [item.lower() for item in parts.split(',')]
all_annotations = list(
annotation.read_annotations(annotations_filename))
annotation.link_up_annotations(all_annotations)
for item in all_annotations:
item.primary = None
annotations = [
item for item in all_annotations if item.type.lower() in types
]
part_annotations = []
seen = set()
queue = [(item, item) for item in annotations]
while queue:
primary, item = queue.pop()
if item.type.lower() in parts:
assert item.primary is None, "Feature with multiple parents"
item.primary = primary
key = (id(primary), item.start, item.end, item.seqid,
item.strand)
# Ignore duplicate exons (many isoforms will have the same exons)
if key not in seen:
seen.add(key)
part_annotations.append(item)
queue.extend((primary, item2) for item2 in item.children)
del seen
del all_annotations
self.log.log('%d annotations\n' % len(annotations))
self.log.log('%d part annotations\n' % len(part_annotations))
#assert annotations, 'No annotations of specified types in file'
for item in part_annotations:
this_extension = self.extension
if "max_extension" in item.attr:
this_extension = min(this_extension,
int(item.attr["max_extension"]))
if item.strand >= 0:
item.tail_pos = item.end
item.end += this_extension
else:
item.tail_pos = item.start
item.start -= this_extension
for item in annotations:
item.hits = [] # [ (tail_length, adaptor_bases) ]
index = span_index.index_annotations(part_annotations)
for alignment in sam.Bam_reader(workspace /
'alignments_filtered_sorted.bam'):
if alignment.is_unmapped or alignment.is_secondary or alignment.is_supplementary:
continue
start = alignment.reference_start
end = alignment.reference_end
alignment_length = end - start
strand = -1 if alignment.flag & sam.FLAG_REVERSE else 1
fragment_feature = annotation.Annotation(
seqid=alignment.reference_name,
start=start,
end=end,
strand=strand)
if strand >= 0:
tail_pos = end
else:
tail_pos = start
tail_length = 0
adaptor_bases = 0
for item in alignment.extra:
if item.startswith('AN:i:'):
tail_length = int(item[5:])
elif item.startswith('AD:i:'):
adaptor_bases = int(item[5:])
hits = index.get(fragment_feature, same_strand=True)
if hits:
gene = min(
hits,
key=lambda gene:
(abs(tail_pos - gene.tail_pos), gene.primary.get_id()))
# Nearest by tail_pos
# failing that, by id to ensure a deterministic choice
gene.primary.hits.append((tail_length, adaptor_bases))
for item in annotations:
del item.parents
del item.children
del item.primary
f = io.open_possibly_compressed_writer(self.prefix + '.pickle.gz')
pickle.dump((workspace.name, workspace.get_tags(), annotations), f,
pickle.HIGHEST_PROTOCOL)
f.close()
def run(self):
#assert not self.utr_only or self.utrs, '--utrs-only yes but no --utrs given'
# Reference genome
#chromosome_lengths = reference_directory.Reference(self.reference, must_exist=True).get_lengths()
chromosomes = collections.OrderedDict(io.read_sequences(
self.reference))
def get_interpeak_seq(peaks):
start = min(item.transcription_stop for item in peaks)
end = max(item.transcription_stop for item in peaks)
if end - start > self.max_seq: return ''
if peaks[0].strand >= 0:
return chromosomes[peaks[0].seqid][start:end]
else:
return bio.reverse_complement(
chromosomes[peaks[0].seqid][start:end])
def get_prepeak_seq(gene, peaks):
if gene.strand >= 0:
start = gene.utr_pos
end = min(item.transcription_stop for item in peaks)
if end - start > self.max_seq: return ''
return chromosomes[gene.seqid][start:end]
else:
start = max(item.transcription_stop for item in peaks)
end = gene.utr_pos
if end - start > self.max_seq: return ''
return bio.reverse_complement(
chromosomes[gene.seqid][start:end])
# Normalization files
if self.norm_file:
norm_file = self.norm_file
else:
nesoni.Norm_from_counts(self.prefix + '-norm', self.counts).run()
norm_file = self.prefix + '-norm.csv'
norms = io.read_grouped_table(norm_file, [('All', str)])['All']
pair_norm_names = []
pair_norms = []
for i in xrange(len(norms)):
pair_norm_names.append(norms.keys()[i] + '-peak1')
pair_norms.append(norms.values()[i])
for i in xrange(len(norms)):
pair_norm_names.append(norms.keys()[i] + '-peak2')
pair_norms.append(norms.values()[i])
io.write_grouped_csv(
self.prefix + '-pairs-norm.csv',
[('All', io.named_list_type(pair_norm_names)(pair_norms))],
comments=['#Normalization'],
)
# Read data
annotations = list(annotation.read_annotations(self.parents))
if self.utrs:
utrs = list(annotation.read_annotations(self.utrs))
else:
utrs = []
children = list(annotation.read_annotations(self.children))
count_table = io.read_grouped_table(self.counts,
[('Count', int),
('Tail_count', int),
('Tail', _float_or_none),
('Proportion', _float_or_none),
('Annotation', str)])
counts = count_table['Count']
tail_counts = count_table['Tail_count']
proportions = count_table['Proportion']
tails = count_table['Tail']
samples = counts.value_type().keys()
sample_tags = {}
for line in count_table.comments:
if line.startswith('#sampleTags='):
parts = line[len('#sampleTags='):].split(',')
assert parts[0] not in sample_tags
sample_tags[parts[0]] = parts
for item in children:
item.weight = sum(counts[item.get_id()][name] *
float(norms[name]['Normalizing.multiplier'])
for name in samples)
parents = []
id_to_parent = {}
for item in annotations:
if item.type != self.parent_type: continue
assert item.get_id(
) not in id_to_parent, 'Duplicate id in parent file: ' + item.get_id(
)
parents.append(item)
id_to_parent[item.get_id()] = item
item.children = []
#item.cds = [ ]
# Default utr
if item.strand >= 0:
item.utr_pos = item.end
else:
item.utr_pos = item.start
if 'three_prime_UTR_start' in item.attr:
if item.strand >= 0:
item.utr_pos = int(item.attr['three_prime_UTR_start']) - 1
else:
item.utr_pos = int(item.attr['three_prime_UTR_start'])
for item in utrs:
assert item.attr[
'Parent'] in id_to_parent, 'Unknown gene ' + item.attr['Parent']
id_to_parent[item.attr['Parent']].utr_pos = (
item.start if item.strand >= 0 else item.end)
for item in children:
item.transcription_stop = item.end if item.strand >= 0 else item.start #End of transcription, 0-based, ie between-positions based
if 'Parent' in item.attr and item.attr.get(
"Relation") != "Antisense":
for item_parent in item.attr['Parent'].split(','):
parent = id_to_parent[item_parent]
parent.children.append(item)
for item in parents:
item.children.sort(key=_annotation_sorter)
relevant = list(item.children)
if self.utr_only:
#if item.strand <= 0:
# relative_utr_start = item.end - int(item.attr['three_prime_UTR_start'])
#else:
# relative_utr_start = int(item.attr['three_prime_UTR_start'])-1 - item.start
#
#def relative_start(peak):
# return item.end-peak.end if item.strand < 0 else peak.start-item.start
#relevant = [ peak for peak in relevant if relative_start(peak) >= relative_utr_start ]
#relevant = [
# peak for peak in relevant
# if (peak.end >= item.utr_pos if item.strand >= 0 else peak.start <= item.utr_pos)
# ]
relevant = [
peak for peak in relevant
if peak.attr.get("Relation") == "3'UTR"
]
if self.top:
relevant.sort(key=lambda peak: peak.weight, reverse=True)
relevant = relevant[:self.top]
relevant.sort(key=_annotation_sorter)
item.relevant_children = relevant
# JSON output
j_data = {}
j_genes = j_data['genes'] = {}
j_genes['__comment__'] = 'start is 0-based'
j_genes['name'] = []
j_genes['chromosome'] = []
j_genes['strand'] = []
j_genes['start'] = []
j_genes['utr'] = []
j_genes['end'] = []
j_genes['gene'] = []
j_genes['product'] = []
j_genes['peaks'] = []
j_genes['relevant_peaks'] = []
#j_genes['cds'] = [ ]
#j_genes['cds_start'] = [ ]
#j_genes['cds_end'] = [ ]
for item in parents:
j_genes['name'].append(item.get_id())
j_genes['chromosome'].append(item.seqid)
j_genes['strand'].append(item.strand)
j_genes['start'].append(item.start)
j_genes['utr'].append(item.utr_pos)
j_genes['end'].append(item.end)
j_genes['gene'].append(
item.attr.get('Name', item.attr.get('gene', '')))
j_genes['product'].append(
item.attr.get('Product', item.attr.get('product', '')))
j_genes['peaks'].append(
[item2.get_id() for item2 in item.children])
j_genes['relevant_peaks'].append(
[item2.get_id() for item2 in item.relevant_children])
#j_genes['cds'].append( item.cds )
#j_genes['cds_start'].append( item.cds_start )
#j_genes['cds_end'].append( item.cds_end )
j_peaks = j_data['peaks'] = {}
j_peaks['__comment__'] = 'start is 0-based'
j_peaks['name'] = []
j_peaks['chromosome'] = []
j_peaks['strand'] = []
j_peaks['start'] = []
j_peaks['end'] = []
j_peaks['parents'] = []
j_peaks['counts'] = []
j_peaks['tail_lengths'] = []
j_peaks['proportion_tailed'] = []
for item in children:
j_peaks['name'].append(item.get_id())
j_peaks['chromosome'].append(item.seqid)
j_peaks['strand'].append(item.strand)
j_peaks['start'].append(item.start)
j_peaks['end'].append(item.end)
j_peaks['parents'].append(item.attr['Parent'].split(',')
if 'Parent' in item.attr else [])
j_peaks['counts'].append(counts[item.get_id()].values())
j_peaks['tail_lengths'].append(
count_table['Tail'][item.get_id()].values())
j_peaks['proportion_tailed'].append(
count_table['Proportion'][item.get_id()].values())
j_samples = j_data['samples'] = {}
j_samples['name'] = []
j_samples['tags'] = []
j_samples['normalizing_multiplier'] = []
for name in samples:
j_samples['name'].append(name)
j_samples['tags'].append(sample_tags[name])
j_samples['normalizing_multiplier'].append(
float(norms[name]['Normalizing.multiplier']))
j_chromosomes = j_data['chromosomes'] = {}
j_chromosomes['name'] = []
j_chromosomes['length'] = []
for name, seq in chromosomes.iteritems():
j_chromosomes['name'].append(name)
j_chromosomes['length'].append(len(seq))
with open(self.prefix + '.json', 'wb') as f:
json.dump(j_data, f)
# Output paired peak file
output_comments = ['#Counts']
output_samples = []
for item in samples:
output_samples.append(item + '-peak1')
output_comments.append('#sampleTags=' +
','.join([item + '-peak1', 'peak1'] +
sample_tags.get(item, [])))
for item in samples:
output_samples.append(item + '-peak2')
output_comments.append('#sampleTags=' +
','.join([item + '-peak2', 'peak2'] +
sample_tags.get(item, [])))
output_names = []
output_counts = []
output_tail_counts = []
output_proportions = []
output_tails = []
output_annotation_fields = [
'gene', 'product', 'biotype', 'mean_tail_1', 'mean_tail_2',
'chromosome', 'strand', 'transcription_stops'
] #, 'interpeak_seq', ]
output_annotations = []
for item in parents:
peaks = item.relevant_children
for i in xrange(len(peaks) - 1):
for j in xrange(i + 1, len(peaks)):
id_i = peaks[i].get_id()
id_j = peaks[j].get_id()
id_pair = item.get_id() + '-' + id_i + '-' + id_j
output_names.append(id_pair)
row = []
row.extend(counts[id_i].values())
row.extend(counts[id_j].values())
output_counts.append(filter(_text, row))
row = []
row.extend(tail_counts[id_i].values())
row.extend(tail_counts[id_j].values())
output_tail_counts.append(filter(_text, row))
row = []
row.extend(proportions[id_i].values())
row.extend(proportions[id_j].values())
output_proportions.append(filter(_text, row))
row = []
row.extend(tails[id_i].values())
row.extend(tails[id_j].values())
output_tails.append(filter(_text, row))
output_annotations.append([
item.attr.get('Name', item.attr.get('gene', '')),
item.attr.get('Product', item.attr.get('product', '')),
item.attr.get('Biotype', ''),
count_table['Annotation'][id_i]['mean-tail'],
count_table['Annotation'][id_j]['mean-tail'],
item.seqid,
str(item.strand),
'%d, %d' % (peaks[i].transcription_stop,
peaks[j].transcription_stop),
#get_interpeak_seq([peaks[i],peaks[j]]),
])
#output_count_table = io.named_matrix_type(output_names,output_samples)(output_counts)
io.write_grouped_csv(
self.prefix + '-pairs.csv',
[
('Count', io.named_matrix_type(output_names,
output_samples)(output_counts)),
('Tail_count',
io.named_matrix_type(output_names,
output_samples)(output_tail_counts)),
('Proportion',
io.named_matrix_type(output_names,
output_samples)(output_proportions)),
('Tail', io.named_matrix_type(output_names,
output_samples)(output_tails)),
('Annotation',
io.named_matrix_type(
output_names,
output_annotation_fields)(output_annotations)),
],
comments=output_comments,
)
def run(self):
annotations = [ ]
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if self.type:
item.type = self.type
annotations.append(item)
annotations.sort(key=lambda item: (item.type, item.seqid, item.strand, item.start))
group = [ ]
groups = [ ]
def emit():
if not group: return
groups.append(group[:])
del group[:]
type = None
seqid = None
strand = None
end = 0
for item in annotations:
if item.type != type or item.seqid != seqid or item.strand != strand or item.start >= end:
emit()
type = item.type
seqid = item.seqid
strand = item.strand
end = item.end-self.overlap
group.append(item)
end = max(item.end-self.overlap, end)
emit()
items = [ ]
id_map = { }
for group in groups:
item = annotation.Annotation()
item.source = group[0].source
item.type = group[0].type
item.seqid = group[0].seqid
item.strand = group[0].strand
item.start = min( item2.start for item2 in group )
item.end = max( item2.end for item2 in group )
item.score = None
item.phase = None
item.attr = { }
for item2 in group:
for key in item2.attr:
if key in item.attr: continue
item.attr[key] = join_descriptions([ item3.attr[key] for item3 in group if key in item3.attr ], self.joiner )
item.parents = [ ]
for item2 in group:
if 'ID' in item2.attr:
assert item2.attr['ID'] not in id_map, 'Duplicate ID: '+item2.attr['ID']
id_map[item2.attr['ID']] = item.attr['ID']
if 'Parent' in item2.attr:
item.parents.append(item2.attr['Parent'])
items.append(item)
for item in items:
if item.parents:
item.attr['Parent'] = join_descriptions([ id_map.get(parent,parent) for parent in item.parents ], ',')
with open(self.prefix+'.gff','wb') as out_file:
annotation.write_gff3_header(out_file)
for item in items:
print >> out_file, item.as_gff()
def run(self):
annotations = []
for filename in self.filenames:
for item in annotation.read_annotations(filename):
if not selection.matches(self.select, [item.type]): continue
if self.type:
item.type = self.type
annotations.append(item)
annotations.sort(
key=lambda item: (item.type, item.seqid, item.strand, item.start))
group = []
groups = []
def emit():
if not group: return
groups.append(group[:])
del group[:]
type = None
seqid = None
strand = None
end = 0
for item in annotations:
if item.type != type or item.seqid != seqid or item.strand != strand or item.start >= end:
emit()
type = item.type
seqid = item.seqid
strand = item.strand
end = item.end - self.overlap
group.append(item)
end = max(item.end - self.overlap, end)
emit()
items = []
id_map = {}
for group in groups:
item = annotation.Annotation()
item.source = group[0].source
item.type = group[0].type
item.seqid = group[0].seqid
item.strand = group[0].strand
item.start = min(item2.start for item2 in group)
item.end = max(item2.end for item2 in group)
item.score = None
item.phase = None
item.attr = {}
for item2 in group:
for key in item2.attr:
if key in item.attr: continue
item.attr[key] = join_descriptions([
item3.attr[key] for item3 in group if key in item3.attr
], self.joiner)
item.parents = []
for item2 in group:
if 'ID' in item2.attr:
assert item2.attr[
'ID'] not in id_map, 'Duplicate ID: ' + item2.attr['ID']
id_map[item2.attr['ID']] = item.attr['ID']
if 'Parent' in item2.attr:
item.parents.append(item2.attr['Parent'])
items.append(item)
for item in items:
if item.parents:
item.attr['Parent'] = join_descriptions(
[id_map.get(parent, parent) for parent in item.parents],
',')
with open(self.prefix + '.gff', 'wb') as out_file:
annotation.write_gff3_header(out_file)
for item in items:
print >> out_file, item.as_gff()
def count_run(
min_score, min_size, max_size, filter_mode, equalize, types, locii,
qualifiers, use_strand, merge_filename, limit, output_prefix, filenames, log):
if filter_mode == 'poly':
use_bam_filename = 'alignments.bam'
use_only_top = True
use_only_monogamous = False
expect_multiple_alignments = True
elif filter_mode == 'mono':
use_bam_filename = 'alignments.bam'
use_only_top = True
use_only_monogamous = True
expect_multiple_alignments = True
else:
assert filter_mode == 'existing', 'Unrecognized filtering mode'
use_bam_filename = 'alignments_filtered.bam'
use_only_top = False
use_only_monogamous = False
expect_multiple_alignments = False
types = types.lower().split(',')
qualifiers = qualifiers.split(',')
if locii:
locii = locii.lower().split(',')
else:
locii = None
assert use_strand is not None, 'You must now explicitly specify --strand'
assert use_strand in ('pool','forward','reverse','both'), "Can't understand --strand specification."
from Bio import Seq, SeqIO
annotation_filenames = [ ]
bam_filenames = [ ]
for arg in filenames:
if annotation.is_annotation_file(arg):
annotation_filenames.append(arg)
else:
bam_filenames.append(arg)
n_samples = len(bam_filenames)
titles = bam_filenames[:]
for i in xrange(len(bam_filenames)):
if os.path.isdir(bam_filenames[i]):
titles[i] = os.path.basename(bam_filenames[i])
if not annotation_filenames:
working = working_directory.Working(bam_filenames[i])
reference_filename = working.get_reference().annotations_filename()
if reference_filename is not None:
annotation_filenames.append(reference_filename)
bam_filenames[i] = os.path.join(bam_filenames[i], use_bam_filename)
assert bam_filenames, 'No reference alignments given'
merge = { }
merge_qualifiers = { }
if merge_filename is not None:
#First line gives qualifiers
#remaining lines give <qualifier> <qualifier...> <gene> <transcript> <transcript...>
f = open(merge_filename,'rU')
qualifiers = f.readline().rstrip('\n').split('\t')
for line in f:
parts = line.rstrip('\n').split('\t')
if not parts: continue
for name in parts[len(qualifiers)+1:]:
assert name not in merge, 'Duplicate feature name in merge file'
merge[name] = parts[len(qualifiers)]
merge_qualifiers[name] = parts[:len(qualifiers)]
f.close()
genes = { } # reference name -> gene index
feature_names = { } # feature_name -> number of occurrences
features = [ ]
n_features = 0
chromosome_length = { }
for filename in bam_filenames:
headers = sam.bam_headers(filename)
for line in headers.split('\n'):
if not line: continue
parts = line.split('\t')
if parts[0] != '@SQ': continue
name = None
length = None
for part in parts[1:]:
if part.startswith('SN:'): name = part[3:]
if part.startswith('LN:'): length = int(part[3:])
assert name is not None and length is not None
if name in chromosome_length:
assert chromosome_length[name] == length
else:
chromosome_length[name] = length
for name in chromosome_length:
genes[name] = span_index.Span_index()
if annotation_filenames:
assert not merge, 'Merging not supported with annotation files'
for filename in annotation_filenames:
for feature in annotation.read_annotations(filename):
if feature.type.lower() not in types: continue
if (locii is not None and
('locus_tag' not in feature.attr or
feature.attr['locus_tag'].lower() not in locii)):
continue
f = Feature(n_samples)
f.name = feature.get_id()
if feature.type.lower() != 'cds' and len(types) > 1:
f.name = feature.type + ':' + f.name
feature_names[f.name] = feature_names.get(f.name,0)+1
if feature_names[f.name] > 1:
f.name += '/%d' % feature_names[f.name]
f.qualifiers = [ feature.attr.get(item,'') for item in qualifiers ]
f.length = feature.end - feature.start
assert feature.seqid in genes, 'Annotation for sequence that is not in BAM files'
genes[feature.seqid].insert(Span_entry(feature.start, feature.end, feature.strand or 1, f))
features.append(f)
else:
# Sequences as features
log.log('No annotation files given or found, using sequences as features\n')
name_feature = { } # (merged)name -> feature
for name in chromosome_length:
merged_name = merge.get(name, name)
if merged_name not in name_feature:
f = Feature(n_samples)
f.name = merged_name
f.length = length
f.qualifiers = merge_qualifiers.get(name, ('',)*len(qualifiers))
n_features += 1
name_feature[merged_name] = f
features.append(f)
else:
f = name_feature[merged_name]
f.length = max(f.length, length) #...
genes[name].insert(Span_entry(0, chromosome_length[name], 1, f))
log.log('%d features\n\n' % len(features))
for name in genes:
genes[name].prepare()
n_fragments = [ 0 ] * n_samples
n_fragments_aligned = [ 0 ] * n_samples
n_low_score = [ 0 ] * n_samples
n_something = [ 0 ] * n_samples
n_multiple = [ 0 ] * n_samples
n_span = [ 0 ] * n_samples
for i in xrange(n_samples):
for read_name, fragment_alignments, unmapped in sam.bam_iter_fragments(bam_filenames[i], 'Counting sample %d of %d' % (i+1,n_samples)):
n_fragments[i] += 1
if not fragment_alignments:
continue
n_fragments_aligned[i] += 1
feature_hits = [ ] # [ [ (feature, strand) ] ]
# Use only top scoring alignments
fragment_scores = [ sum( al.get_AS() for al in item ) for item in fragment_alignments ]
best_score = max(fragment_scores)
if min_score is not None and best_score < min_score:
n_low_score[i] += 1
continue
if use_only_top:
cutoff = max(best_score, min_score)
else:
cutoff = min_score
fragment_alignments = [ item
for item, score in zip(fragment_alignments, fragment_scores)
if score >= cutoff ]
for alignments in fragment_alignments:
strand = -1 if alignments[0].flag&sam.FLAG_REVERSE else 1
start = min(item.pos-1 for item in alignments)
end = max(item.pos+item.length-1 for item in alignments)
length = end-start
if min_size is not None and length < min_size: continue
if max_size is not None and length > max_size: continue
rname = alignments[0].rname
strand = -1 if alignments[0].flag&sam.FLAG_REVERSE else 1
assert alignments[0].rname in genes, 'Alignment refers to sequence not present in GENBANK file'
this_feature_hits = [ ]
for item in genes[rname].get(start, end):
rel_strand = strand * item.strand
key = (item.feature, rel_strand)
if key in this_feature_hits: continue
this_feature_hits.append( key )
if not use_only_monogamous or len(fragment_alignments) == 1:
item.feature.count[rel_strand][i] += 1
if this_feature_hits:
feature_hits.append( this_feature_hits )
if len(this_feature_hits) > 1:
for a in this_feature_hits:
for b in this_feature_hits:
if a[0] is b[0]: continue
a[0].common[(a[1],b[1])][b[0]] += 1
if len(feature_hits) > 0:
n_something[i] += 1
#else:
# print fragment_alignments
# print genes[fragment_alignments[0][0].rname].indexes
# print
if len(feature_hits) > 1:
n_multiple[i] += 1
for j in xrange(len(feature_hits)):
for k in xrange(len(feature_hits)):
if j == k: continue
for a in feature_hits[j]:
for b in feature_hits[k]:
if a[0] is b[0]: continue
a[0].ambiguous[(a[1],b[1])][b[0]] += 1
if any(len(item) > 1 for item in feature_hits): n_span[i] += 1
if limit is not None and n_fragments[i] >= limit: break
grace.status('')
#log.log('%s\n' % titles[i])
#log.log('%20s fragments\n' % grace.pretty_number(n_fragments[i]))
#log.log('%20s fragments aligned to the reference\n' % grace.pretty_number(n_fragments_aligned[i]))
#if n_low_score[i]:
# log.log('%20s had too low an alignment score, discarded\n' % grace.pretty_number(n_low_score[i]))
#log.log('%20s aligned to an annotated gene\n' % grace.pretty_number(n_something[i]))
#if expect_multiple_alignments or n_multiple[i]:
# log.log('%20s aligned to multiple genes\n' % grace.pretty_number(n_multiple[i]))
#log.log('%20s had an alignment that spanned multiple genes\n' % grace.pretty_number(n_span[i]))
#log.log('\n')
log.datum(titles[i], 'fragments', n_fragments[i])
log.datum(titles[i], 'fragments aligned to the reference', n_fragments_aligned[i])
if n_low_score[i]:
log.datum(titles[i], 'had too low an alignment score, discarded', n_low_score[i])
log.datum(titles[i], 'aligned to an annotated gene', n_something[i])
if expect_multiple_alignments or n_multiple[i]:
log.datum(titles[i], 'aligned to multiple genes', n_multiple[i])
log.datum(titles[i],'had an alignment that spanned multiple genes', n_span[i])
log.log('\n')
strandedness = [ ]
for feature in features:
n_forward = sum(feature.count[1])
n_reverse = sum(feature.count[-1])
if n_forward+n_reverse < 5: continue
strandedness.append( (n_forward-n_reverse)*100.0 / (n_forward+n_reverse) )
strandedness = sum(strandedness) / len(strandedness)
log.log('Strand specificity: %.0f%%\n'
' (~ -100%% reverse strand, ~ 0%% non-specific, ~ 100%% forward strand\n'
' Average over all features with at least 5 hits.)\n'
% strandedness)
if use_strand == 'pool':
getters = [ lambda f: (feature.name,
add_lists(feature.count[1],feature.count[-1]),
add_defdicts(feature.common[(1,1)],
feature.common[(1,-1)],
feature.common[(-1,1)],
feature.common[(-1,-1)]),
add_defdicts(feature.ambiguous[(1,1)],
feature.ambiguous[(1,-1)],
feature.ambiguous[(-1,1)],
feature.ambiguous[(-1,-1)])) ]
elif use_strand == 'forward':
getters = [ lambda f: (feature.name, feature.count[1], feature.common[(1,1)], feature.ambiguous[(1,1)]) ]
elif use_strand == 'reverse':
getters = [ lambda f: (feature.name, feature.count[-1], feature.common[(-1,-1)], feature.ambiguous[(-1,-1)]) ]
elif use_strand == 'both':
getters = [ lambda f: (feature.name, feature.count[1], feature.common[(1,1)], feature.ambiguous[(1,1)]),
lambda f: (feature.name + 'r', feature.count[-1], feature.common[(-1,-1)], feature.ambiguous[(-1,-1)]) ]
total_hits = [0] * n_samples
for feature in features:
for getter in getters:
total_hits = add_lists(total_hits, getter(feature)[1])
if equalize:
min_hits = min(total_hits)
p = [ float(min_hits)/item for item in total_hits ]
total_hits = [ min_hits ] * n_samples
f = open(output_prefix + '.txt', 'wb')
#log.attach(open(output_prefix + '_log.txt', 'wb'))
print >> f, tab_encode(
[ 'Feature' ] +
titles +
[ 'RPKM ' + item for item in titles ] +
[ 'Length' ] +
qualifiers +
[ 'On same fragment' ] +
([ 'Ambiguous alignment' ] if expect_multiple_alignments else [ ])
)
for feature in features:
for getter in getters:
feature_name, count, common, ambiguous = getter(feature)
if equalize:
count = [
subsample(count[i], p[i])
for i in xrange(n_samples)
]
rpkm = [ count[i] * 1.0e9 / feature.length / total_hits[i] for i in xrange(n_samples) ]
common_str = ' '.join(
'%dx%s' % (item[1],item[0])
for item in sorted(common.items(), key=lambda item:item[1], reverse=True)
)
ambiguous_str = ' '.join(
'%dx%s' % (item[1],item[0])
for item in sorted(ambiguous.items(), key=lambda item:item[1], reverse=True)
)
print >> f, tab_encode(
[ feature_name ] +
[ str(item) for item in count ] +
[ '%.2f' % item for item in rpkm ] +
[ str(feature.length) ] +
list(feature.qualifiers) +
[ common_str ] +
([ ambiguous_str ] if expect_multiple_alignments else [ ])
)
f.close()
def run(self):
work = self.get_workspace()
work.update_param(remove=['tail_tools_reference_version'])
nesoni.Make_reference(
self.output_dir,
filenames = self.filenames,
snpeff = False,
cs = 'ifavailable' if self.index else False,
ls = False,
bowtie = 'ifavailable' if self.index else False,
).run()
annotations = list(annotation.read_annotations(work/'reference.gff'))
annotation.link_up_annotations(annotations)
with open(work/'utr.gff','wb') as f:
annotation.write_gff3_header(f)
for gene in annotations:
if gene.type != 'gene': continue
mrnas = [ item for item in gene.children if item.type == 'mRNA' ]
utr_5primes = [ ]
for mrna in mrnas:
cdss = [ item for item in mrna.children if item.type == 'CDS' ]
exons = [ item for item in mrna.children if item.type == 'exon' ]
if not cdss or not exons: continue
if gene.strand >= 0:
cds_3prime = max(item.end for item in cdss)
for item in exons:
if item.end > cds_3prime:
utr_5primes.append(max(item.start,cds_3prime))
else:
cds_3prime = min(item.start for item in cdss)
for item in exons:
if item.start < cds_3prime:
utr_5primes.append(min(item.end,cds_3prime))
if gene.strand >= 0:
utr_start = max(utr_5primes) if utr_5primes else gene.end
utr_end = max(utr_start+1,gene.end)
else:
utr_end = min(utr_5primes) if utr_5primes else gene.start
utr_start = min(gene.start,utr_end-1)
attr = gene.attr.copy()
attr['Parent'] = attr['ID']
attr['ID'] = attr['ID']+'-3UTR'
thing = annotation.Annotation(
source = 'tt',
type = 'three_prime_utr',
seqid = gene.seqid,
strand = gene.strand,
start = utr_start,
end = utr_end,
attr = attr,
)
print >> f, thing.as_gff()
work.update_param(tail_tools_reference_version=work.VERSION)
def run(self):
items = list(annotation.read_annotations(self.parent))
annotation.link_up_annotations(items)
for item in items:
assert len(item.parents) <= 1
genes = [ item for item in items if item.type == "gene" ]
downstrand_genes = [ _extend(_three_prime(item), self.extension) for item in genes ]
exons = [ item for item in items if item.type == "exon" ]
utrs = [ _extend(item, self.extension) for item in items if item.type == "three_prime_utr" ]
gene_index = span_index.index_annotations(genes)
downstrand_gene_index = span_index.index_annotations(downstrand_genes)
exon_index = span_index.index_annotations(exons)
utr_index = span_index.index_annotations(utrs)
peaks = [ ]
for peak in annotation.read_annotations(self.child):
if float(peak.attr.get("mean_tail","0.0")) < self.min_tail:
continue
peaks.append(peak)
for peak in peaks:
# Query is final base in genome before poly(A) starts
query = peak.three_prime().shifted(-1,0)
hit_to = "3'UTR"
hits = [ item.parents[0].parents[0] for item in
utr_index.get(query, True) ]
if not hits:
hit_to = "Exon"
hits = [ item.parents[0].parents[0] for item in
exon_index.get(query, True) ]
# For non-coding RNAs, which don't have a 3' UTR
if not hits:
hit_to = "Downstrand"
hits = downstrand_gene_index.get(query, True)
if not hits:
hit_to = "Intron"
hits = gene_index.get(query, True)
antisense_hits = gene_index.get(query.reversed(), True)
if not hits:
hit_to = "Antisense"
hits = antisense_hits
if hits:
peak.attr["Parent"] = join_descriptions([ item.get_id() for item in hits ], ",")
peak.attr["Relation"] = hit_to
peak.attr["Name"] = join_descriptions([ item.attr.get("Name","") for item in hits ])
peak.attr["Product"] = hit_to + " " + join_descriptions([ item.attr.get("Product","") for item in hits ])
peak.attr["Biotype"] = join_descriptions([ item.attr.get("Biotype","") for item in hits ])
if antisense_hits:
peak.attr["Antisense_parent"] = join_descriptions([ item.get_id() for item in antisense_hits ], ",")
peak.attr["Antisense_name"] = join_descriptions([ item.attr.get("Name","") for item in antisense_hits ])
peak.attr["Antisense_product"] = "Antisense " + join_descriptions([ item.attr.get("Product","") for item in antisense_hits ])
peak.attr["Antisense_biotype"] = join_descriptions([ item.attr.get("Biotype","") for item in antisense_hits])
annotation.write_gff3(self.prefix+"-parent.gff", genes) #Hmm
annotation.write_gff3(self.prefix+"-child.gff", peaks)
def run(self):
assert self.extension is not None, '--extension must be specified'
#workspace = self.get_workspace()
workspace = working_directory.Working(self.working_dir, must_exist=True)
if self.annotations == None:
reference = workspace.get_reference()
annotations_filename = reference.annotations_filename()
else:
annotations_filename = self.annotations
types = [ item.lower() for item in self.types.split(',') ]
parts = self.parts or self.types
parts = [ item.lower() for item in parts.split(',') ]
all_annotations = list(annotation.read_annotations(annotations_filename))
annotation.link_up_annotations(all_annotations)
for item in all_annotations:
item.primary = None
annotations = [
item
for item in all_annotations
if item.type.lower() in types
]
part_annotations = [ ]
seen = set()
queue = [ (item,item) for item in annotations ]
while queue:
primary, item = queue.pop()
if item.type.lower() in parts:
assert item.primary is None, "Feature with multiple parents"
item.primary = primary
key = (id(primary),item.start,item.end,item.seqid,item.strand)
# Ignore duplicate exons (many isoforms will have the same exons)
if key not in seen:
seen.add(key)
part_annotations.append(item)
queue.extend( (primary, item2) for item2 in item.children )
del seen
del all_annotations
self.log.log('%d annotations\n' % len(annotations))
self.log.log('%d part annotations\n' % len(part_annotations))
#assert annotations, 'No annotations of specified types in file'
for item in part_annotations:
this_extension = self.extension
if "max_extension" in item.attr:
this_extension = min(this_extension,int(item.attr["max_extension"]))
if item.strand >= 0:
item.tail_pos = item.end
item.end += this_extension
else:
item.tail_pos = item.start
item.start -= this_extension
for item in annotations:
item.hits = [] # [ (tail_length, adaptor_bases) ]
index = span_index.index_annotations(part_annotations)
for alignment in sam.Bam_reader(workspace/'alignments_filtered_sorted.bam'):
if alignment.is_unmapped or alignment.is_secondary or alignment.is_supplementary:
continue
start = alignment.reference_start
end = alignment.reference_end
alignment_length = end-start
strand = -1 if alignment.flag&sam.FLAG_REVERSE else 1
fragment_feature = annotation.Annotation(
seqid=alignment.reference_name,
start=start,
end=end,
strand=strand
)
if strand >= 0:
tail_pos = end
else:
tail_pos = start
tail_length = 0
adaptor_bases = 0
for item in alignment.extra:
if item.startswith('AN:i:'):
tail_length = int(item[5:])
elif item.startswith('AD:i:'):
adaptor_bases = int(item[5:])
hits = index.get(fragment_feature, same_strand=True)
if hits:
gene = min(hits, key=lambda gene:
(abs(tail_pos - gene.tail_pos), gene.primary.get_id()))
# Nearest by tail_pos
# failing that, by id to ensure a deterministic choice
gene.primary.hits.append( (tail_length,adaptor_bases) )
for item in annotations:
del item.parents
del item.children
del item.primary
f = io.open_possibly_compressed_writer(self.prefix + '.pickle.gz')
pickle.dump((workspace.name, workspace.get_tags(), annotations), f, pickle.HIGHEST_PROTOCOL)
f.close()
def run(self):
items = list(annotation.read_annotations(self.parent))
annotation.link_up_annotations(items)
for item in items:
assert len(item.parents) <= 1
genes = [ item for item in items if item.type == "gene" ]
downstrand_genes = [ _extend(_three_prime(item), self.extension) for item in genes ]
exons = [ item for item in items if item.type == "exon" ]
utrs = [ _extend(item, self.extension) for item in items if item.type == "three_prime_utr" ]
gene_index = span_index.index_annotations(genes)
downstrand_gene_index = span_index.index_annotations(downstrand_genes)
exon_index = span_index.index_annotations(exons)
utr_index = span_index.index_annotations(utrs)
peaks = list(annotation.read_annotations(self.child))
for peak in peaks:
# Query is final base in genome before poly(A) starts
query = peak.three_prime().shifted(-1,0)
hit_to = "3'UTR"
hits = [ item.parents[0].parents[0] for item in
utr_index.get(query, True) ]
if not hits:
hit_to = "Exon"
hits = [ item.parents[0].parents[0] for item in
exon_index.get(query, True) ]
# For non-coding RNAs, which don't have a 3' UTR
if not hits:
hit_to = "Downstrand"
hits = downstrand_gene_index.get(query, True)
if not hits:
hit_to = "Intron"
hits = gene_index.get(query, True)
antisense_hits = gene_index.get(query.reversed(), True)
if not hits:
hit_to = "Antisense"
hits = antisense_hits
if hits:
peak.attr["Parent"] = join_descriptions([ item.get_id() for item in hits ], ",")
peak.attr["Relation"] = hit_to
peak.attr["Name"] = join_descriptions([ item.attr.get("Name","") for item in hits ])
peak.attr["Product"] = hit_to + " " + join_descriptions([ item.attr.get("Product","") for item in hits ])
peak.attr["Biotype"] = join_descriptions([ item.attr.get("Biotype","") for item in hits ])
if antisense_hits:
peak.attr["Antisense_parent"] = join_descriptions([ item.get_id() for item in antisense_hits ], ",")
peak.attr["Antisense_name"] = join_descriptions([ item.attr.get("Name","") for item in antisense_hits ])
peak.attr["Antisense_product"] = "Antisense " + join_descriptions([ item.attr.get("Product","") for item in antisense_hits ])
peak.attr["Antisense_biotype"] = join_descriptions([ item.attr.get("Biotype","") for item in antisense_hits])
annotation.write_gff3(self.prefix+"-parent.gff", genes) #Hmm
annotation.write_gff3(self.prefix+"-child.gff", peaks)
def run(self):
names = [ sample.output_dir for sample in self.samples ]
#os.path.splitext(os.path.split(item)[1])[0]
#for item in self.reads
#]
reference = reference_directory.Reference(self.reference, must_exist=True)
workspace = io.Workspace(self.output_dir, must_exist=False)
samplespace = io.Workspace(workspace/'samples', must_exist=False)
plotspace = io.Workspace(workspace/'plots', must_exist=False)
expressionspace = io.Workspace(workspace/'expression', must_exist=False)
testspace = io.Workspace(workspace/'test', must_exist=False)
testspace_dedup = io.Workspace(workspace/'test-dedup', must_exist=False)
file_prefix = self.file_prefix
if file_prefix and not file_prefix.endswith('-'):
file_prefix += '-'
#dirs = [
# workspace/item
# for item in names
#]
samples = [ ]
for sample in self.samples:
samples.append(sample(
samplespace / sample.output_dir,
reference = self.reference,
))
dirs = [ item.output_dir for item in samples ]
polya_dirs = [ item + '-polyA' for item in dirs ]
interleaved = [ item2 for item in zip(dirs,polya_dirs) for item2 in item ]
clipper_logs = [ join(item.output_dir, 'clipped_reads_log.txt') for item in samples ]
filter_logs = [ join(item.output_dir, 'filter_log.txt') for item in samples ]
filter_polya_logs = [ join(item.output_dir + '-polyA', 'filter_log.txt') for item in samples ]
#filter_logs = [ item.get_filter_action().log_filename() for item in samples ]
#filter_polya_logs = [ item.get_polya_filter_action().log_filename() for item in samples ]
analyse_template = tail_lengths.Analyse_tail_counts(
working_dirs = dirs,
saturation = 0,
extension = self.extension,
annotations = reference/'reference.gff',
types = 'gene',
)
with nesoni.Stage() as stage:
for item in samples:
item.process_make(stage)
nesoni.Norm_from_samples(
workspace/'norm',
working_dirs = dirs
).make()
def writer():
for row in io.read_table(workspace/'norm.csv'):
row['Name'] = row['Name']+'-polyA'
yield row
io.write_csv(workspace/'norm-polyA.csv', writer(), comments=['Normalization'])
with nesoni.Stage() as stage:
if self.include_plots:
for plot_name, directories, norm_filename in [
('all', dirs, workspace/'norm.csv'),
('polyA', polya_dirs, workspace/'norm-polyA.csv'),
]:
nesoni.IGV_plots(
plotspace/plot_name,
working_dirs = directories,
label_prefix = plot_name+' ',
raw = True,
norm = True,
genome = reference.get_genome_filename(),
norm_file = norm_filename,
#delete_igv = False,
).process_make(stage)
analyse_gene_counts_0 = analyse_template(
output_dir = expressionspace/'genewise',
saturation = 0,
extension = self.extension,
title = 'Genewise expression - ' + self.title,
file_prefix = file_prefix+'genewise-',
)
analyse_gene_counts_0.process_make(stage)
analyse_gene_counts_1 = analyse_template(
output_dir = expressionspace/'genewise-dedup',
saturation = 1,
title = 'Genewise expression with read deduplication - ' + self.title,
file_prefix = file_prefix+'genewise-dedup-',
)
analyse_gene_counts_1.process_make(stage)
stage.process(self._run_peaks,
workspace=workspace, expressionspace=expressionspace, reference=reference,
polya_dirs=polya_dirs, analyse_template=analyse_template, file_prefix=file_prefix,
)
with nesoni.Stage() as stage:
for test in self.tests:
test(
output_dir = testspace/test.output_dir,
analysis = self.output_dir
).process_make(stage)
test(
output_dir = testspace_dedup/test.output_dir,
analysis = self.output_dir,
dedup = True,
).process_make(stage)
#===============================================
# Report
#===============================================
r = reporting.Reporter(os.path.join(self.output_dir, 'report'), self.title, self.file_prefix)
r.heading('Alignment to reference')
r.report_logs('alignment-statistics',
#[ workspace/'stats.txt' ] +
clipper_logs + filter_logs + #filter_polya_logs +
[ expressionspace/('genewise','aggregate-tail-counts_log.txt') ],
filter=lambda sample, field: (
field not in [
'fragments','fragments aligned to the reference','reads kept',
'average depth of coverage, ambiguous',
'average depth of coverage, unambiguous',
]
),
)
if self.include_plots:
r.heading('IGV plots')
r.p('These files show the depth of coverage. They can be viewed with the IGV genome browser.')
genome_files = [ ]
if self.include_genome:
genome_files.append(reference.get_genome_filename())
genome_dir = reference.get_genome_dir()
base = os.path.split(self.genome_dir)[1]
for filename in os.listdir(genome_dir):
genome_files.append((
os.path.join(genome_dir, filename),
os.path.join(base, filename)
))
r.p(r.tar('igv-plots',
genome_files +
glob.glob(plotspace/'*.tdf')
))
if self.include_bams:
r.heading('BAM files')
r.p('These BAM files contain the alignments of reads to the reference sequences.')
r.p('Reads with a poly(A) tail have an \'AN\' attribute giving the length of non-templated poly(A) sequence. '
'Tail-tools only treats a read as having a tail if this length is at least 4.')
bam_files = [ ]
for name in names:
bam_files.append( (samplespace/(name,'alignments_filtered_sorted.bam'),name+'.bam') )
bam_files.append( (samplespace/(name,'alignments_filtered_sorted.bam.bai'),name+'.bam.bai') )
r.p(r.tar('bam-files', bam_files))
r.heading('Genewise expression')
io.symbolic_link(source=expressionspace/('genewise','report'),link_name=r.workspace/'genewise')
r.p('<a href="genewise/index.html">→ Genewise expression</a>')
io.symbolic_link(source=expressionspace/('genewise-dedup','report'),link_name=r.workspace/'genewise-dedup')
r.p('<a href="genewise-dedup/index.html">→ Genewise expression with read deduplication</a>')
r.heading('Peakwise expression')
web.Geneview_webapp(r.workspace/'view').run()
peak_filename = expressionspace/('peakwise','features-with-data.gff')
n_peaks = len(list(annotation.read_annotations(peak_filename)))
r.p('%d peaks called (%d poly(A) reads were required to call a peak).' % (n_peaks, self.peak_min_depth))
r.p(r.get(peak_filename, name='peaks.gff') + ' - peaks called')
#if self.groups:
#r.subheading('Peak shift between groups')
#r.p(r.get(workspace/('peak-shift','grouped.csv')) + ' - genes with a potential peak shift')
#r.get(workspace/('peak-shift','grouped.json'))
#r.subheading('Peak shift between samples')
#r.p(r.get(workspace/('peak-shift','individual.csv')) + ' - genes with a potential peak shift')
#r.get(workspace/('peak-shift','individual.json'))
io.symbolic_link(source=expressionspace/('peakwise','report'),link_name=r.workspace/'peakwise')
r.p('<a href="peakwise/index.html">→ Peakwise expression</a>')
io.symbolic_link(source=expressionspace/('peakwise-dedup','report'),link_name=r.workspace/'peakwise-dedup')
r.p('<a href="peakwise-dedup/index.html">→ Peakwise expression with read deduplication</a>')
if self.tests:
r.heading('Differential tests')
for test in self.tests:
io.symbolic_link(source=testspace/test.output_dir,link_name=r.workspace/('test-'+test.output_dir))
io.symbolic_link(source=testspace_dedup/test.output_dir,link_name=r.workspace/('test-dedup-'+test.output_dir))
r.p('<a href="test-%s">→ %s</a> '
' <a href="test-dedup-%s" style="font-size: 66%%">[→ Deduplicated version]</a>' % (test.output_dir, test.get_title(), test.output_dir))
r.heading('Gene viewers')
r.p('Having identified interesting genes from heatmaps and differential tests above, '
'these viewers allow specific genes to be examined in detail.')
if self.groups:
r.p('<a href="view.html?json=%sgrouped.json">→ Gene viewer, grouped samples</a>' % r.file_prefix)
r.p('<a href="view.html?json=%sindividual.json">→ Gene viewer, individual samples</a>' % r.file_prefix)
r.write('<p/><hr>\n')
r.p('Note: Use deduplicated versions with care. '
'They may possibly provide more significant results, however they are less quantitative. '
'Read deduplication involves throwing away a large amount of data, much of which will not be a technical artifact. '
'Deduplicated versions might best be viewed as a check on data quality.')
r.p('This set of genes was used in the analysis:')
r.p(r.get(reference/'reference.gff') + ' - Reference annotations in GFF3 format')
r.p(r.get(reference/'utr.gff') + ' - 3\' UTR regions')
r.p('tail-tools version '+tail_tools.VERSION)
r.p('nesoni version '+nesoni.VERSION)
#r.p('SHRiMP version '+grace.get_shrimp_2_version())
r.close()
def run(self):
work = self.get_workspace()
work.update_param(remove=['tail_tools_reference_version'])
nesoni.Make_reference(
self.output_dir,
filenames=self.filenames,
snpeff=False,
cs='ifavailable' if self.index else False,
ls=False,
bowtie='ifavailable' if self.index else False,
).run()
annotations = list(annotation.read_annotations(work / 'reference.gff'))
annotation.link_up_annotations(annotations)
exon_index = span_index.index_annotations(
[item for item in annotations if item.type == "exon"])
mrna_end_index = span_index.index_annotations([
item.three_prime() for item in annotations if item.type == "mRNA"
])
mrna_utrs = []
gene_utrs = []
for gene in annotations:
if gene.type != 'gene': continue
mrnas = [item for item in gene.children if item.type == 'mRNA']
assert mrnas, "Gene without any mRNAs: " + gene.get_id()
gene.attr['color'] = '#880088'
gene.start = min(item.start for item in mrnas)
gene.end = max(item.end for item in mrnas)
gene.attr["max_extension"] = str(
_max_extension(gene, exon_index, mrna_end_index))
gene_utr_5primes = []
for mrna in mrnas:
assert mrna.strand == gene.strand, mrna
assert mrna.seqid == gene.seqid, mrna
mrna.attr["max_extension"] = str(
_max_extension(mrna, exon_index, mrna_end_index))
cdss = [item for item in mrna.children if item.type == 'CDS']
exons = [item for item in mrna.children if item.type == 'exon']
if not exons: continue
#link up annotations sorts children, so final is really final
for item in exons[:-1]:
item.attr["max_extension"] = "0"
exons[-1].attr["max_extension"] = mrna.attr["max_extension"]
if not cdss: continue
mrna_utr_5primes = []
if gene.strand >= 0:
cds_3prime = max(item.end for item in cdss)
for item in exons:
if item.end >= cds_3prime:
mrna_utr_5primes.append(max(
item.start, cds_3prime))
else:
cds_3prime = min(item.start for item in cdss)
for item in exons:
if item.start <= cds_3prime:
mrna_utr_5primes.append(min(item.end, cds_3prime))
if mrna.strand >= 0:
utr_start = min(
mrna_utr_5primes) if mrna_utr_5primes else mrna.end
utr_end = max(utr_start + 1, mrna.end)
gene_utr_5primes.append(utr_start)
else:
utr_end = max(
mrna_utr_5primes) if mrna_utr_5primes else mrna.start
utr_start = min(mrna.start, utr_end - 1)
gene_utr_5primes.append(utr_end)
attr = mrna.attr.copy()
attr['Parent'] = attr['ID']
attr['ID'] = attr['ID'] + '-3UTR'
attr['color'] = '#008888'
utr = annotation.Annotation(
source='tt',
type='three_prime_utr',
seqid=mrna.seqid,
strand=mrna.strand,
start=utr_start,
end=utr_end,
attr=attr,
)
max_ext = _max_extension(utr, exon_index, mrna_end_index)
utr.attr["max_extension"] = str(max_ext)
#Only include if there is an annotated 3' UTR or end is not in the middle of some other isoform's exon
if utr_end - utr_start + max_ext > 1:
mrna_utrs.append(utr)
if gene.strand >= 0:
utr_start = max(
gene_utr_5primes) if gene_utr_5primes else gene.end
utr_end = max(utr_start + 1, gene.end)
else:
utr_end = min(
gene_utr_5primes) if gene_utr_5primes else gene.start
utr_start = min(gene.start, utr_end - 1)
attr = gene.attr.copy()
attr['Parent'] = attr['ID']
attr['ID'] = attr['ID'] + '-3UTR'
attr['color'] = '#008888'
utr = annotation.Annotation(
source='tt',
type='three_prime_utr',
seqid=gene.seqid,
strand=gene.strand,
start=utr_start,
end=utr_end,
attr=attr,
)
utr.attr["max_extension"] = str(
_max_extension(utr, exon_index, mrna_end_index))
gene_utrs.append(utr)
annotation.write_gff3(work / 'reference.gff', annotations + mrna_utrs)
annotation.write_gff3(work / 'utr.gff', gene_utrs)
work.update_param(tail_tools_reference_version=work.VERSION)
def count_run(min_score, min_size, max_size, filter_mode, equalize, types,
locii, qualifiers, use_strand, merge_filename, limit,
output_prefix, filenames, log):
if filter_mode == 'poly':
use_bam_filename = 'alignments.bam'
use_only_top = True
use_only_monogamous = False
expect_multiple_alignments = True
elif filter_mode == 'mono':
use_bam_filename = 'alignments.bam'
use_only_top = True
use_only_monogamous = True
expect_multiple_alignments = True
else:
assert filter_mode == 'existing', 'Unrecognized filtering mode'
use_bam_filename = 'alignments_filtered.bam'
use_only_top = False
use_only_monogamous = False
expect_multiple_alignments = False
types = types.lower().split(',')
qualifiers = qualifiers.split(',')
if locii:
locii = locii.lower().split(',')
else:
locii = None
assert use_strand is not None, 'You must now explicitly specify --strand'
assert use_strand in ('pool', 'forward', 'reverse',
'both'), "Can't understand --strand specification."
from Bio import Seq, SeqIO
annotation_filenames = []
bam_filenames = []
for arg in filenames:
if annotation.is_annotation_file(arg):
annotation_filenames.append(arg)
else:
bam_filenames.append(arg)
n_samples = len(bam_filenames)
titles = bam_filenames[:]
for i in xrange(len(bam_filenames)):
if os.path.isdir(bam_filenames[i]):
titles[i] = os.path.basename(bam_filenames[i])
if not annotation_filenames:
working = working_directory.Working(bam_filenames[i])
reference_filename = working.get_reference(
).annotations_filename()
if reference_filename is not None:
annotation_filenames.append(reference_filename)
bam_filenames[i] = os.path.join(bam_filenames[i], use_bam_filename)
assert bam_filenames, 'No reference alignments given'
merge = {}
merge_qualifiers = {}
if merge_filename is not None:
#First line gives qualifiers
#remaining lines give <qualifier> <qualifier...> <gene> <transcript> <transcript...>
f = open(merge_filename, 'rU')
qualifiers = f.readline().rstrip('\n').split('\t')
for line in f:
parts = line.rstrip('\n').split('\t')
if not parts: continue
for name in parts[len(qualifiers) + 1:]:
assert name not in merge, 'Duplicate feature name in merge file'
merge[name] = parts[len(qualifiers)]
merge_qualifiers[name] = parts[:len(qualifiers)]
f.close()
genes = {} # reference name -> gene index
feature_names = {} # feature_name -> number of occurrences
features = []
n_features = 0
chromosome_length = {}
for filename in bam_filenames:
headers = sam.bam_headers(filename)
for line in headers.split('\n'):
if not line: continue
parts = line.split('\t')
if parts[0] != '@SQ': continue
name = None
length = None
for part in parts[1:]:
if part.startswith('SN:'): name = part[3:]
if part.startswith('LN:'): length = int(part[3:])
assert name is not None and length is not None
if name in chromosome_length:
assert chromosome_length[name] == length
else:
chromosome_length[name] = length
for name in chromosome_length:
genes[name] = span_index.Span_index()
if annotation_filenames:
assert not merge, 'Merging not supported with annotation files'
for filename in annotation_filenames:
for feature in annotation.read_annotations(filename):
if feature.type.lower() not in types: continue
if (locii is not None and
('locus_tag' not in feature.attr
or feature.attr['locus_tag'].lower() not in locii)):
continue
f = Feature(n_samples)
f.name = feature.get_id()
if feature.type.lower() != 'cds' and len(types) > 1:
f.name = feature.type + ':' + f.name
feature_names[f.name] = feature_names.get(f.name, 0) + 1
if feature_names[f.name] > 1:
f.name += '/%d' % feature_names[f.name]
f.qualifiers = [
feature.attr.get(item, '') for item in qualifiers
]
f.length = feature.end - feature.start
assert feature.seqid in genes, 'Annotation for sequence that is not in BAM files'
genes[feature.seqid].insert(
Span_entry(feature.start, feature.end, feature.strand or 1,
f))
features.append(f)
else:
# Sequences as features
log.log(
'No annotation files given or found, using sequences as features\n'
)
name_feature = {} # (merged)name -> feature
for name in chromosome_length:
merged_name = merge.get(name, name)
if merged_name not in name_feature:
f = Feature(n_samples)
f.name = merged_name
f.length = length
f.qualifiers = merge_qualifiers.get(name,
('', ) * len(qualifiers))
n_features += 1
name_feature[merged_name] = f
features.append(f)
else:
f = name_feature[merged_name]
f.length = max(f.length, length) #...
genes[name].insert(Span_entry(0, chromosome_length[name], 1, f))
log.log('%d features\n\n' % len(features))
for name in genes:
genes[name].prepare()
n_fragments = [0] * n_samples
n_fragments_aligned = [0] * n_samples
n_low_score = [0] * n_samples
n_something = [0] * n_samples
n_multiple = [0] * n_samples
n_span = [0] * n_samples
for i in xrange(n_samples):
for read_name, fragment_alignments, unmapped in sam.bam_iter_fragments(
bam_filenames[i],
'Counting sample %d of %d' % (i + 1, n_samples)):
n_fragments[i] += 1
if not fragment_alignments:
continue
n_fragments_aligned[i] += 1
feature_hits = [] # [ [ (feature, strand) ] ]
# Use only top scoring alignments
fragment_scores = [
sum(al.get_AS() for al in item) for item in fragment_alignments
]
best_score = max(fragment_scores)
if min_score is not None and best_score < min_score:
n_low_score[i] += 1
continue
if use_only_top:
cutoff = max(best_score, min_score)
else:
cutoff = min_score
fragment_alignments = [
item
for item, score in zip(fragment_alignments, fragment_scores)
if score >= cutoff
]
for alignments in fragment_alignments:
strand = -1 if alignments[0].flag & sam.FLAG_REVERSE else 1
start = min(item.pos - 1 for item in alignments)
end = max(item.pos + item.length - 1 for item in alignments)
length = end - start
if min_size is not None and length < min_size: continue
if max_size is not None and length > max_size: continue
rname = alignments[0].rname
strand = -1 if alignments[0].flag & sam.FLAG_REVERSE else 1
assert alignments[
0].rname in genes, 'Alignment refers to sequence not present in GENBANK file'
this_feature_hits = []
for item in genes[rname].get(start, end):
rel_strand = strand * item.strand
key = (item.feature, rel_strand)
if key in this_feature_hits: continue
this_feature_hits.append(key)
if not use_only_monogamous or len(
fragment_alignments) == 1:
item.feature.count[rel_strand][i] += 1
if this_feature_hits:
feature_hits.append(this_feature_hits)
if len(this_feature_hits) > 1:
for a in this_feature_hits:
for b in this_feature_hits:
if a[0] is b[0]: continue
a[0].common[(a[1], b[1])][b[0]] += 1
if len(feature_hits) > 0:
n_something[i] += 1
#else:
# print fragment_alignments
# print genes[fragment_alignments[0][0].rname].indexes
# print
if len(feature_hits) > 1:
n_multiple[i] += 1
for j in xrange(len(feature_hits)):
for k in xrange(len(feature_hits)):
if j == k: continue
for a in feature_hits[j]:
for b in feature_hits[k]:
if a[0] is b[0]: continue
a[0].ambiguous[(a[1], b[1])][b[0]] += 1
if any(len(item) > 1 for item in feature_hits): n_span[i] += 1
if limit is not None and n_fragments[i] >= limit: break
grace.status('')
#log.log('%s\n' % titles[i])
#log.log('%20s fragments\n' % grace.pretty_number(n_fragments[i]))
#log.log('%20s fragments aligned to the reference\n' % grace.pretty_number(n_fragments_aligned[i]))
#if n_low_score[i]:
# log.log('%20s had too low an alignment score, discarded\n' % grace.pretty_number(n_low_score[i]))
#log.log('%20s aligned to an annotated gene\n' % grace.pretty_number(n_something[i]))
#if expect_multiple_alignments or n_multiple[i]:
# log.log('%20s aligned to multiple genes\n' % grace.pretty_number(n_multiple[i]))
#log.log('%20s had an alignment that spanned multiple genes\n' % grace.pretty_number(n_span[i]))
#log.log('\n')
log.datum(titles[i], 'fragments', n_fragments[i])
log.datum(titles[i], 'fragments aligned to the reference',
n_fragments_aligned[i])
if n_low_score[i]:
log.datum(titles[i], 'had too low an alignment score, discarded',
n_low_score[i])
log.datum(titles[i], 'aligned to an annotated gene', n_something[i])
if expect_multiple_alignments or n_multiple[i]:
log.datum(titles[i], 'aligned to multiple genes', n_multiple[i])
log.datum(titles[i], 'had an alignment that spanned multiple genes',
n_span[i])
log.log('\n')
strandedness = []
for feature in features:
n_forward = sum(feature.count[1])
n_reverse = sum(feature.count[-1])
if n_forward + n_reverse < 5: continue
strandedness.append(
(n_forward - n_reverse) * 100.0 / (n_forward + n_reverse))
strandedness = sum(strandedness) / len(strandedness)
log.log(
'Strand specificity: %.0f%%\n'
' (~ -100%% reverse strand, ~ 0%% non-specific, ~ 100%% forward strand\n'
' Average over all features with at least 5 hits.)\n' % strandedness)
if use_strand == 'pool':
getters = [
lambda f:
(feature.name, add_lists(feature.count[1], feature.count[-1]),
add_defdicts(feature.common[(1, 1)], feature.common[
(1, -1)], feature.common[(-1, 1)], feature.common[(-1, -1)]),
add_defdicts(feature.ambiguous[(1, 1)], feature.ambiguous[
(1, -1)], feature.ambiguous[(-1, 1)], feature.ambiguous[
(-1, -1)]))
]
elif use_strand == 'forward':
getters = [
lambda f: (feature.name, feature.count[1], feature.common[
(1, 1)], feature.ambiguous[(1, 1)])
]
elif use_strand == 'reverse':
getters = [
lambda f: (feature.name, feature.count[-1], feature.common[
(-1, -1)], feature.ambiguous[(-1, -1)])
]
elif use_strand == 'both':
getters = [
lambda f: (feature.name, feature.count[1], feature.common[
(1, 1)], feature.ambiguous[(1, 1)]), lambda f:
(feature.name + 'r', feature.count[-1], feature.common[
(-1, -1)], feature.ambiguous[(-1, -1)])
]
total_hits = [0] * n_samples
for feature in features:
for getter in getters:
total_hits = add_lists(total_hits, getter(feature)[1])
if equalize:
min_hits = min(total_hits)
p = [float(min_hits) / item for item in total_hits]
total_hits = [min_hits] * n_samples
f = open(output_prefix + '.txt', 'wb')
#log.attach(open(output_prefix + '_log.txt', 'wb'))
print >> f, tab_encode(
['Feature'] + titles + ['RPKM ' + item for item in titles] +
['Length'] + qualifiers + ['On same fragment'] +
(['Ambiguous alignment'] if expect_multiple_alignments else []))
for feature in features:
for getter in getters:
feature_name, count, common, ambiguous = getter(feature)
if equalize:
count = [subsample(count[i], p[i]) for i in xrange(n_samples)]
rpkm = [
count[i] * 1.0e9 / feature.length / total_hits[i]
for i in xrange(n_samples)
]
common_str = ' '.join(
'%dx%s' % (item[1], item[0]) for item in sorted(
common.items(), key=lambda item: item[1], reverse=True))
ambiguous_str = ' '.join(
'%dx%s' % (item[1], item[0]) for item in sorted(
ambiguous.items(), key=lambda item: item[1], reverse=True))
print >> f, tab_encode(
[feature_name] + [str(item) for item in count] +
['%.2f' % item for item in rpkm] + [str(feature.length)] +
list(feature.qualifiers) + [common_str] +
([ambiguous_str] if expect_multiple_alignments else []))
f.close()
def run(self):
#assert not self.utr_only or self.utrs, '--utrs-only yes but no --utrs given'
# Reference genome
#chromosome_lengths = reference_directory.Reference(self.reference, must_exist=True).get_lengths()
chromosomes = collections.OrderedDict(io.read_sequences(self.reference))
def get_interpeak_seq(peaks):
start = min(item.transcription_stop for item in peaks)
end = max(item.transcription_stop for item in peaks)
if end-start > self.max_seq: return ''
if peaks[0].strand >= 0:
return chromosomes[peaks[0].seqid][start:end]
else:
return bio.reverse_complement(chromosomes[peaks[0].seqid][start:end])
def get_prepeak_seq(gene,peaks):
if gene.strand >= 0:
start = gene.utr_pos
end = min(item.transcription_stop for item in peaks)
if end-start > self.max_seq: return ''
return chromosomes[gene.seqid][start:end]
else:
start = max(item.transcription_stop for item in peaks)
end = gene.utr_pos
if end-start > self.max_seq: return ''
return bio.reverse_complement(chromosomes[gene.seqid][start:end])
# Normalization files
if self.norm_file:
norm_file = self.norm_file
else:
nesoni.Norm_from_counts(self.prefix+'-norm', self.counts).run()
norm_file = self.prefix+'-norm.csv'
norms = io.read_grouped_table(norm_file, [('All',str)])['All']
pair_norm_names = [ ]
pair_norms = [ ]
for i in xrange(len(norms)):
pair_norm_names.append(norms.keys()[i]+'-peak1')
pair_norms.append(norms.values()[i])
for i in xrange(len(norms)):
pair_norm_names.append(norms.keys()[i]+'-peak2')
pair_norms.append(norms.values()[i])
io.write_grouped_csv(
self.prefix+'-pairs-norm.csv',
[('All',io.named_list_type(pair_norm_names)(pair_norms))],
comments=['#Normalization'],
)
# Read data
annotations = list(annotation.read_annotations(self.parents))
if self.utrs:
utrs = list(annotation.read_annotations(self.utrs))
else:
utrs = [ ]
children = list(annotation.read_annotations(self.children))
count_table = io.read_grouped_table(self.counts, [
('Count',int),
('Tail_count',int),
('Tail',_float_or_none),
('Proportion',_float_or_none),
('Annotation',str)
])
counts = count_table['Count']
tail_counts = count_table['Tail_count']
proportions = count_table['Proportion']
tails = count_table['Tail']
samples = counts.value_type().keys()
sample_tags = { }
for line in count_table.comments:
if line.startswith('#sampleTags='):
parts = line[len('#sampleTags='):].split(',')
assert parts[0] not in sample_tags
sample_tags[parts[0]] = parts
for item in children:
item.weight = sum( counts[item.get_id()][name] * float(norms[name]['Normalizing.multiplier']) for name in samples )
parents = [ ]
id_to_parent = { }
for item in annotations:
if item.type != self.parent_type: continue
assert item.get_id() not in id_to_parent, 'Duplicate id in parent file: '+item.get_id()
parents.append(item)
id_to_parent[item.get_id()] = item
item.children = [ ]
#item.cds = [ ]
# Default utr
if item.strand >= 0:
item.utr_pos = item.end
else:
item.utr_pos = item.start
if 'three_prime_UTR_start' in item.attr:
if item.strand >= 0:
item.utr_pos = int(item.attr['three_prime_UTR_start'])-1
else:
item.utr_pos = int(item.attr['three_prime_UTR_start'])
for item in utrs:
assert item.attr['Parent'] in id_to_parent, 'Unknown gene '+item.attr['Parent']
id_to_parent[item.attr['Parent']].utr_pos = (item.start if item.strand >= 0 else item.end)
for item in children:
item.transcription_stop = item.end if item.strand >= 0 else item.start #End of transcription, 0-based, ie between-positions based
if 'Parent' in item.attr:
for item_parent in item.attr['Parent'].split(','):
parent = id_to_parent[item_parent]
parent.children.append(item)
for item in parents:
item.children.sort(key=_annotation_sorter)
relevant = list(item.children)
if self.utr_only:
#if item.strand <= 0:
# relative_utr_start = item.end - int(item.attr['three_prime_UTR_start'])
#else:
# relative_utr_start = int(item.attr['three_prime_UTR_start'])-1 - item.start
#
#def relative_start(peak):
# return item.end-peak.end if item.strand < 0 else peak.start-item.start
#relevant = [ peak for peak in relevant if relative_start(peak) >= relative_utr_start ]
relevant = [
peak for peak in relevant
if (peak.end >= item.utr_pos if item.strand >= 0 else peak.start <= item.utr_pos)
]
if self.top:
relevant.sort(key=lambda peak:peak.weight, reverse=True)
relevant = relevant[:self.top]
relevant.sort(key=_annotation_sorter)
item.relevant_children = relevant
# JSON output
j_data = { }
j_genes = j_data['genes'] = { }
j_genes['__comment__'] = 'start is 0-based'
j_genes['name'] = [ ]
j_genes['chromosome'] = [ ]
j_genes['strand'] = [ ]
j_genes['start'] = [ ]
j_genes['utr'] = [ ]
j_genes['end'] = [ ]
j_genes['gene'] = [ ]
j_genes['product'] = [ ]
j_genes['peaks'] = [ ]
j_genes['relevant_peaks'] = [ ]
#j_genes['cds'] = [ ]
#j_genes['cds_start'] = [ ]
#j_genes['cds_end'] = [ ]
for item in parents:
j_genes['name'].append( item.get_id() )
j_genes['chromosome'].append( item.seqid )
j_genes['strand'].append( item.strand )
j_genes['start'].append( item.start )
j_genes['utr'].append( item.utr_pos )
j_genes['end'].append( item.end )
j_genes['gene'].append( item.attr.get('Name',item.attr.get('gene','')) )
j_genes['product'].append( item.attr.get('Product',item.attr.get('product','')) )
j_genes['peaks'].append( [ item2.get_id() for item2 in item.children ] )
j_genes['relevant_peaks'].append( [ item2.get_id() for item2 in item.relevant_children ] )
#j_genes['cds'].append( item.cds )
#j_genes['cds_start'].append( item.cds_start )
#j_genes['cds_end'].append( item.cds_end )
j_peaks = j_data['peaks'] = { }
j_peaks['__comment__'] = 'start is 0-based'
j_peaks['name'] = [ ]
j_peaks['chromosome'] = [ ]
j_peaks['strand'] = [ ]
j_peaks['start'] = [ ]
j_peaks['end'] = [ ]
j_peaks['parents'] = [ ]
j_peaks['counts'] = [ ]
j_peaks['tail_lengths'] = [ ]
j_peaks['proportion_tailed'] = [ ]
for item in children:
j_peaks['name'].append( item.get_id() )
j_peaks['chromosome'].append( item.seqid )
j_peaks['strand'].append( item.strand )
j_peaks['start'].append( item.start )
j_peaks['end'].append( item.end )
j_peaks['parents'].append( item.attr['Parent'].split(',') if 'Parent' in item.attr else [ ])
j_peaks['counts'].append( counts[item.get_id()].values() )
j_peaks['tail_lengths'].append( count_table['Tail'][item.get_id()].values() )
j_peaks['proportion_tailed'].append( count_table['Proportion'][item.get_id()].values() )
j_samples = j_data['samples'] = { }
j_samples['name'] = [ ]
j_samples['tags'] = [ ]
j_samples['normalizing_multiplier'] = [ ]
for name in samples:
j_samples['name'].append(name)
j_samples['tags'].append(sample_tags[name])
j_samples['normalizing_multiplier'].append(float(norms[name]['Normalizing.multiplier']))
j_chromosomes = j_data['chromosomes'] = { }
j_chromosomes['name'] = [ ]
j_chromosomes['length'] = [ ]
for name, seq in chromosomes.iteritems():
j_chromosomes['name'].append(name)
j_chromosomes['length'].append(len(seq))
with open(self.prefix + '.json','wb') as f:
json.dump(j_data, f)
# Output paired peak file
output_comments = [ '#Counts' ]
output_samples = [ ]
for item in samples:
output_samples.append(item+'-peak1')
output_comments.append('#sampleTags=' + ','.join([item+'-peak1','peak1']+sample_tags.get(item,[])))
for item in samples:
output_samples.append(item+'-peak2')
output_comments.append('#sampleTags=' + ','.join([item+'-peak2','peak2']+sample_tags.get(item,[])))
output_names = [ ]
output_counts = [ ]
output_tail_counts = [ ]
output_proportions = [ ]
output_tails = [ ]
output_annotation_fields = [ 'gene', 'product', 'mean_tail_1', 'mean_tail_2', 'chromosome', 'strand',
'transcription_stops' ] #, 'interpeak_seq', ]
output_annotations = [ ]
for item in parents:
peaks = item.relevant_children
for i in xrange(len(peaks)-1):
for j in xrange(i+1, len(peaks)):
id_i = peaks[i].get_id()
id_j = peaks[j].get_id()
id_pair = item.get_id() + '-'+id_i+'-'+id_j
output_names.append(id_pair)
row = [ ]
row.extend(counts[id_i].values())
row.extend(counts[id_j].values())
output_counts.append(filter(_text,row))
row = [ ]
row.extend(tail_counts[id_i].values())
row.extend(tail_counts[id_j].values())
output_tail_counts.append(filter(_text,row))
row = [ ]
row.extend(proportions[id_i].values())
row.extend(proportions[id_j].values())
output_proportions.append(filter(_text,row))
row = [ ]
row.extend(tails[id_i].values())
row.extend(tails[id_j].values())
output_tails.append(filter(_text,row))
output_annotations.append([
item.attr.get('Name',item.attr.get('gene','')),
item.attr.get('Product',item.attr.get('product','')),
count_table['Annotation'][id_i]['mean-tail'],
count_table['Annotation'][id_j]['mean-tail'],
item.seqid,
str(item.strand),
'%d, %d' % (peaks[i].transcription_stop,peaks[j].transcription_stop),
#get_interpeak_seq([peaks[i],peaks[j]]),
])
#output_count_table = io.named_matrix_type(output_names,output_samples)(output_counts)
io.write_grouped_csv(
self.prefix + '-pairs.csv',
[
('Count',io.named_matrix_type(output_names,output_samples)(output_counts)),
('Tail_count',io.named_matrix_type(output_names,output_samples)(output_tail_counts)),
('Proportion',io.named_matrix_type(output_names,output_samples)(output_proportions)),
('Tail',io.named_matrix_type(output_names,output_samples)(output_tails)),
('Annotation',io.named_matrix_type(output_names,output_annotation_fields)(output_annotations)),
],
comments=output_comments,
)
# # Chi Sq tests
#
# #for id in relation:
# # peaks = relation[id]
# # if len(peaks) < 2: continue
#
# mats = [ ]
# genes = [ ]
# products = [ ]
# mean_tails = [ ]
# prop_tails = [ ]
#
# peak_names = [ ]
# chromosome_names = [ ]
# strands = [ ]
# transcription_stops = [ ]
# interpeak_seqs = [ ]
# prepeak_seqs = [ ]
#
# for parent in parents:
# id = parent.get_id()
# peaks = parent.relevant_children
# if len(peaks) < 2: continue
#
# matrix = [ ]
# for item in peaks:
# matrix.append(counts[item.get_id()].values())
#
# mats.append(
# runr.R_literal(id) + ' = ' +
# runr.R_literal(matrix)
# )
#
# genes.append(parent.attr.get('Name',parent.attr.get('gene','')))
# products.append(parent.attr.get('Product',parent.attr.get('product','')))
#
# def format_mean(s):
# if s == 'NA': return 'NA'
# return '%.1f' % float(s)
# mean_tails.append(', '.join( format_mean(count_table['Annotation'][item.get_id()]['mean-tail']) for item in peaks ))
#
# def format_prop(s):
# if s == 'NA': return 'NA'
# return '%.2f' % float(s)
# prop_tails.append(', '.join( format_prop(count_table['Annotation'][item.get_id()]['proportion-with-tail']) for item in peaks ))
#
# peak_names.append(', '.join(item.get_id() for item in peaks))
# chromosome_names.append(parent.seqid)
# strands.append(parent.strand)
# transcription_stops.append(', '.join(str(item.transcription_stop) for item in peaks))
# interpeak_seqs.append(get_interpeak_seq(peaks))
# prepeak_seqs.append(get_prepeak_seq(parent,peaks))
#
# #if len(mats) >= 10: break
#
# text = 'cat("Loading data into R+\n")\n'
# text += 'data <- list(\n' + ',\n'.join(mats) + ')\n'
# text += CHISQ
#
# runr.run_script(text,
# OUTPUT_FILENAME=self.prefix+'.csv',
# GENES = genes,
# PRODUCTS = products,
# MEAN_TAILS = mean_tails,
# PROP_TAILS = prop_tails,
# PEAK_NAMES = peak_names,
# CHROMOSOME_NAMES = chromosome_names,
# STRANDS = strands,
# TRANSCRIPTION_STOPS = transcription_stops,
# INTERPEAK_SEQS = interpeak_seqs,
# PREPEAK_SEQS = prepeak_seqs,
# )
#
def run(self):
assert self.extension is not None, '--extension must be specified'
#workspace = self.get_workspace()
workspace = working_directory.Working(self.working_dir, must_exist=True)
if self.annotations == None:
reference = workspace.get_reference()
annotations_filename = reference.annotations_filename()
else:
annotations_filename = self.annotations
types = [ item.lower() for item in self.types.split(',') ]
annotations = [
item
for item in annotation.read_annotations(annotations_filename)
if item.type.lower() in types
]
self.log.log('%d annotations\n' % len(annotations))
assert annotations, 'No annotations of specified types in file'
index = { }
for item in annotations:
if item.strand >= 0:
item.tail_pos = item.end
item.end += self.extension
else:
item.tail_pos = item.start
item.start -= self.extension
if item.seqid not in index:
index[item.seqid] = span_index.Span_index()
index[item.seqid].insert(item)
item.hits = [] # [ (rel_start, rel_end, tail_length) ]
for item in index.itervalues():
item.prepare()
for read_name, fragment_alignments, unmapped in sam.bam_iter_fragments(workspace/'alignments_filtered.bam'):
for fragment in fragment_alignments:
start = min(item.pos-1 for item in fragment)
end = max(item.pos+item.length-1 for item in fragment)
alignment_length = end-start
strand = -1 if fragment[0].flag&sam.FLAG_REVERSE else 1
if strand >= 0:
tail_pos = end
else:
tail_pos = start
tail_length = 0
adaptor_bases = 0
for item in fragment[0].extra:
if item.startswith('AN:i:'):
tail_length = int(item[5:])
elif item.startswith('AD:i:'):
adaptor_bases = int(item[5:])
if fragment[0].rname in index:
hits = [
gene
for gene in index[fragment[0].rname].get(start,end)
if gene.strand == strand
]
if hits:
gene = min(hits, key=lambda gene: (abs(tail_pos - gene.tail_pos), gene.get_id()))
# Nearest by tail_pos
# failing that, by id to ensure a deterministic choice
if strand > 0:
rel_start = start - gene.start
rel_end = end - gene.start
else:
rel_start = gene.end - end
rel_end = gene.end - start
gene.hits.append( (rel_start,rel_end,tail_length,adaptor_bases) )
f = io.open_possibly_compressed_writer(self.prefix + '.pickle.gz')
pickle.dump((workspace.name, workspace.get_tags(), annotations), f, pickle.HIGHEST_PROTOCOL)
f.close()
