def draw(subnetwork, filename):
try:
# Numpy array format
row = subnetwork[:, 0]
col = subnetwork[:, 1]
data = subnetwork[:, 2]
except TypeError:
# Scipy sparse format
row = subnetwork.row
col = subnetwork.col
data = subnetwork.data
row_indices = stats.rankdata(
np.concatenate((row, col)), method="dense"
)
col_indices = stats.rankdata(
np.concatenate((col, row)), method="dense"
)
data = np.concatenate((data, data))
# print("Row length: {}, col length: {}, data length: {}"
# "".format(len(row_indices), len(col_indices), len(data)))
unique_row = np.unique(row)
unique_col = np.unique(col)
# print("Network shape: {},{}".format(len(unique_row),
# len(unique_col)))
size = len(np.unique(np.concatenate((unique_row, unique_col)))) + 1
# print("Size of matrix to draw: {}".format(size))
try:
sparse_subnet = sparse.coo_matrix(
(data, (row_indices, col_indices)), shape=(size, size)
)
binning_factor = (size // max_size_matrix) + 1
binned_subnet = hcs.bin_sparse(
sparse_subnet, subsampling_factor=binning_factor
)
dense_subnet = binned_subnet.todense()
diagonal = np.diag(np.diag(dense_subnet))
normed_subnet = hcs.normalize_dense(dense_subnet - diagonal)
vmax = np.percentile(normed_subnet, saturation_threshold)
spaceless_pdf_plot_maker(normed_subnet, filename, vmax=vmax)
except MemoryError:
logger.warning(
"Warning, couldn't save matrix due to memory issues"
)
def retrieve_reads_contig_wise(sam_merged, contig_data, output_dir):
contig_dict = dict()
opened_files = dict()
def close_all_files():
for my_file in opened_files.values():
my_file.close()
with open("contig_data.txt") as contig_data:
for line in contig_data:
fields = line.split()
node = fields[0]
core = fields[-3]
contig_dict[node] = core
query_getter = operator.attrgetter("query_name")
with pysam.AlignmentFile(sam_merged, "rb") as sam_handle:
for (my_read_name,
alignment_pool) in itertools.groupby(sam_handle, query_getter):
my_read_set = dict()
my_core_set = set()
while "Reading alignments from alignment pool":
try:
my_alignment = next(alignment_pool)
# print(contig_dict[my_alignment.reference_name])
is_reverse = my_alignment.is_reverse
my_seq = my_alignment.query_sequence
my_qual = my_alignment.query_qualities
my_qual_string = pysam.array_to_qualitystring(my_qual)
if is_reverse:
my_seq_string = str(Seq(my_seq).reverse_complement())
my_qual_string = my_qual_string[::-1]
else:
my_seq_string = my_seq
my_seq_tuple = (my_seq_string, my_qual_string)
if len(my_read_set) > 2:
logger.warning(
"Something's gone wrong with read set %s, as "
"there are %s of them",
my_read_name,
len(my_read_set),
)
elif len(my_read_set) == 0:
my_read_set[my_seq_tuple] = "forward"
elif my_seq_tuple not in my_read_set.keys():
my_read_set[my_seq_tuple] = "reverse"
try:
ref = contig_dict[my_alignment.reference_name]
my_core_set.add(ref)
except KeyError:
my_core_set.add("unknown")
except StopIteration:
if len(my_read_set) == 2:
for core_name in my_core_set:
for my_tuple, file_id in my_read_set.items():
if file_id == "forward":
file_end = ".end1"
elif file_id == "reverse":
file_end = ".end2"
basename = "{core_name}{file_end}".format(
core_name, file_end)
filename = os.path.join(output_dir, basename)
try:
file_to_write = opened_files[filename]
except KeyError:
file_to_write = open(filename, "w")
opened_files[filename] = file_to_write
except IOError:
logger.error(
"Error when trying to handle"
"%s. Maybe there are too many opened"
"files at once: %s",
filename,
len(opened_files),
)
raise
seq, qual = my_tuple
line = "@{}\n{}\n+\n{}\n".format(
my_read_name, seq, qual)
file_to_write.write(line)
elif len(my_read_set) == 1:
for core_name in my_core_set:
file_end = ".end"
basename = "{}{}".format(core_name, file_end)
filename = os.path.join(output_dir, basename)
try:
file_to_write = opened_files[filename]
except KeyError:
file_to_write = open(filename, "w")
opened_files[filename] = file_to_write
except IOError:
logger.error(
"Error when trying to handle"
"%s. Maybe there are too many opened"
"files at once: %s",
filename,
len(opened_files),
)
raise
seq, qual = my_read_set.keys()[0]
line = "@{}\n{}\n+\n{}\n".format(
my_read_name, seq, qual)
file_to_write.write(line)
else:
logger.warning(
"Something's gone wrong with read set %s, as "
"there are %s of them",
my_read_name,
len(my_read_set),
)
break
close_all_files()
def alignment_to_contacts(
sam_merged,
assembly,
output_dir,
output_file_network=DEFAULT_NETWORK_FILE_NAME,
output_file_chunk_data=DEFAULT_CHUNK_DATA_FILE_NAME,
parameters=DEFAULT_PARAMETERS,
):
"""Generates a network file (in edgelist form) from an
alignment in sam or bam format. Contigs are virtually split into
'chunks' of nearly fixed size (by default between 500 and 1000 bp)
to reduce size bias. The chunks are the network nodes and the edges
are the contact counts.
The network is in a strict barebone form so that it can be reused and
imported quickly into other applications etc. Verbose information about
every single node in the network is written on a 'chunk data' file,
by default called 'idx_contig_hit_size_cov.txt'
Parameters
----------
sam_merged : file, str or pathlib.Path
The alignment file in SAM/BAM format to be processed.
assembly : file, str or pathlib.Path
The initial assembly acting as the alignment file's reference genome.
output_dir : str or pathlib.Path
The output directory to write the network and chunk data into.
output_dir_file_network : str or pathlib.Path, optional
The specific file name for the output network file. Default is
network.txt
output_file_chunk_data : str or pathlib.Path, optional
The specific file name for the output chunk data file. Default is
idx_contig_hit_size_cov.txt
parameters : dict, optional
A dictionary of parameters for converting the alignment file into a
network. These are:
-size_chunk_threshold: the size (in bp) under which chunks are
discarded. Default is 500.
-mapq_threshold: the mapping quality under which alignments are
discarded. Default is 10.
-chunk_size: the default chunk size (in bp) when applicable, save
smaller contigs or tail-ends. Default is 1000.
-read_size: the size of reads used for mapping. Default is 65.
-self_contacts: whether to count alignments between a chunk and
itself. Default is False.
-normalized: whether to normalize contacts by their coverage.
Default is False.
Returns
-------
chunk_complete_data : dict
A dictionary where the keys are chunks in (contig, position) form and
the values are their id, name, total contact count, size and coverage.
all_contacts : dict
A counter dictionary where the keys are chunk pairs and the values are
their contact count.
"""
all_contacts = collections.Counter()
all_chunks = collections.Counter()
# Initialize parameters
chunk_size = int(parameters["chunk_size"])
mapq_threshold = int(parameters["mapq_threshold"])
size_chunk_threshold = int(parameters["size_chunk_threshold"])
read_size = int(parameters["read_size"])
self_contacts = parameters["self_contacts"]
normalized = parameters["normalized"]
logger.info("Establishing chunk list...")
chunk_complete_data = dict()
# Get all information about all chunks from all contigs
# (this gets updated at the end)
global_id = 1
for record in SeqIO.parse(assembly, "fasta"):
length = len(record.seq)
n_chunks = length // chunk_size
n_chunks += (length % chunk_size) >= size_chunk_threshold
for i in range(n_chunks):
if (i + 1) * chunk_size <= length:
size = chunk_size
else:
size = length % chunk_size
chunk_name = "{}_{}".format(record.id, i)
chunk_complete_data[chunk_name] = {
"id": global_id,
"hit": 0,
"size": size,
"coverage": 0,
}
global_id += 1
logger.info("Opening alignment files...")
current_read = None
# Read the BAM file to detect contacts.
with pysam.AlignmentFile(sam_merged, "rb") as alignment_merged_handle:
names = alignment_merged_handle.references
lengths = alignment_merged_handle.lengths
names_and_lengths = {
name: length
for name, length in zip(names, lengths)
}
logger.info("Reading contacts...")
# Since the BAM file is supposed to be sorted and interleaved,
# pairs should be always grouped with one below the other (the exact
# order doesn't matter since the network is symmetric, so we simply
# treat the first one as 'forward' and the second one as 'reverse')
# We keep iterating until two consecutive reads have the same name,
# discarding ones that don't.
while "Reading forward and reverse alignments alternatively":
try:
my_read = next(alignment_merged_handle)
if current_read is None:
# First read
current_read = my_read
continue
elif current_read.query_name != my_read.query_name:
# print("{}_{}".format(current_read, my_read))
current_read = my_read
continue
read_forward, read_reverse = current_read, my_read
except StopIteration:
break
# Get a bunch of info about the alignments to pass the tests below
read_name_forward = read_forward.query_name
read_name_reverse = read_reverse.query_name
flag_forward, flag_reverse = read_forward.flag, read_reverse.flag
try:
assert read_name_forward == read_name_reverse
except AssertionError:
logger.error(
"Reads don't have the same name: "
"%s and %s",
read_name_forward,
read_name_reverse,
)
raise
# To check if a flag contains 4
# (digit on the third position from the right in base 2),
# 4 = unmapped in SAM spec
def is_unmapped(flag):
return np.base_repr(flag, padding=3)[-3] == "1"
if is_unmapped(flag_forward) or is_unmapped(flag_reverse):
# print("Detected unmapped read on one end, skipping")
continue
contig_name_forward = read_forward.reference_name
contig_name_reverse = read_reverse.reference_name
len_contig_for = names_and_lengths[contig_name_forward]
len_contig_rev = names_and_lengths[contig_name_reverse]
position_forward = read_forward.reference_start
position_reverse = read_reverse.reference_start
mapq_forward = read_forward.mapping_quality
mapq_reverse = read_reverse.mapping_quality
# Some more tests: checking for size, map quality, map status etc.
mapq_test = min(mapq_forward, mapq_reverse) > mapq_threshold
min_length = min(len_contig_for, len_contig_rev)
length_test = min_length > size_chunk_threshold
# Trickest test:
#
#
# contig
# pos1 pos2
# ^ ^
# |-------|-------|-------|-------|---|
# <-------><------><------><------><--> <->
# chunk chunk tail size_chunk_threshold
#
# Test is passed if tail >= size_chunk_threshold (pos2)
# or if the position is a non-tail chunk (pos1)
if position_forward < chunk_size * (len_contig_for // chunk_size):
current_chunk_forward_size = chunk_size
else:
current_chunk_forward_size = len_contig_for % chunk_size
if position_reverse < chunk_size * (len_contig_rev // chunk_size):
current_chunk_reverse_size = chunk_size
else:
current_chunk_reverse_size = len_contig_rev % chunk_size
min_chunk_size = min(current_chunk_forward_size,
current_chunk_reverse_size)
chunk_test = min_chunk_size >= size_chunk_threshold
if mapq_test and length_test and chunk_test:
chunk_forward = position_forward // chunk_size
chunk_reverse = position_reverse // chunk_size
chunk_name_forward = "{}_{}".format(contig_name_forward,
chunk_forward)
chunk_name_reverse = "{}_{}".format(contig_name_reverse,
chunk_reverse)
if self_contacts or chunk_name_forward != chunk_name_reverse:
contact = tuple(
sorted((chunk_name_forward, chunk_name_reverse)))
all_contacts[contact] += 1
chunk_key_forward = (
chunk_name_forward,
current_chunk_forward_size,
)
all_chunks[chunk_key_forward] += 1
chunk_key_reverse = (
chunk_name_reverse,
current_chunk_reverse_size,
)
all_chunks[chunk_key_reverse] += 1
logger.info("Writing chunk data...")
# Now we can update the chunk dictionary
# with the info we gathered from the BAM file
output_chunk_data_path = os.path.join(output_dir, output_file_chunk_data)
with open(output_chunk_data_path, "w") as chunk_data_file_handle:
for name in sorted(chunk_complete_data.keys()):
chunk_data = chunk_complete_data[name]
size = chunk_data["size"]
chunk = (name, chunk_data["size"])
hit = all_chunks[chunk]
coverage = hit * read_size * 1.0 / size
try:
chunk_complete_data[name]["hit"] = hit
chunk_complete_data[name]["coverage"] = coverage
except KeyError:
logger.error("A mismatch was detected between the reference "
"genome and the genome used for the alignment "
"file, some sequence names were not found")
raise
idx = chunk_complete_data[name]["id"]
line = "{}\t{}\t{}\t{}\t{}\n".format(idx, name, hit, size,
coverage)
chunk_data_file_handle.write(line)
# Lastly, generate the network proper
logger.info("Writing network...")
output_network_path = os.path.join(output_dir, output_file_network)
with open(output_network_path, "w") as network_file_handle:
for chunks in sorted(all_contacts.keys()):
chunk_name1, chunk_name2 = chunks
contact_count = all_contacts[chunks]
if normalized:
coverage1 = chunk_complete_data[chunk_name1]["coverage"]
coverage2 = chunk_complete_data[chunk_name2]["coverage"]
mean_coverage = np.sqrt(coverage1 * coverage2)
effective_count = contact_count * 1.0 / mean_coverage
else:
effective_count = contact_count
try:
idx1 = chunk_complete_data[chunk_name1]["id"]
idx2 = chunk_complete_data[chunk_name2]["id"]
line = "{}\t{}\t{}\n".format(idx1, idx2, effective_count)
network_file_handle.write(line)
except KeyError as e:
logger.warning("Mismatch detected: %s", e)
return chunk_complete_data, all_contacts