From f1fe040e026ec002573a16339b80848d38aeb173 Mon Sep 17 00:00:00 2001
From: Mia Croiset <mia.croiset@ens-lyon.fr>
Date: Fri, 10 Feb 2023 09:54:32 +0100
Subject: [PATCH] TEST fragment enzyme modules
---
bin/__init__.py | 1 +
bin/hicstuff_digest.py | 489 ++++++++
bin/hicstuff_fragments.py | 40 +
bin/hicstuff_io.py | 1379 +++++++++++++++++++++
bin/hicstuff_log.py | 89 ++
conf/hicstuff.config | 31 +
modules/local/hicstuff/fragment_enzyme.nf | 21 +
subworkflows/local/hicstuff_sub.nf | 12 +
workflows/hicstuff.nf | 8 +-
9 files changed, 2067 insertions(+), 3 deletions(-)
create mode 100644 bin/__init__.py
create mode 100644 bin/hicstuff_digest.py
create mode 100755 bin/hicstuff_fragments.py
create mode 100644 bin/hicstuff_io.py
create mode 100644 bin/hicstuff_log.py
create mode 100644 modules/local/hicstuff/fragment_enzyme.nf
diff --git a/bin/__init__.py b/bin/__init__.py
new file mode 100644
index 0000000..813219d
--- /dev/null
+++ b/bin/__init__.py
@@ -0,0 +1 @@
+from .bin import *
diff --git a/bin/hicstuff_digest.py b/bin/hicstuff_digest.py
new file mode 100644
index 0000000..3fba3da
--- /dev/null
+++ b/bin/hicstuff_digest.py
@@ -0,0 +1,489 @@
+#!/usr/bin/env python3
+# coding: utf-8
+
+"""Genome digestion
+Functions used to write auxiliary instagraal compatible
+sparse matrices.
+"""
+
+from Bio import SeqIO, SeqUtils
+from Bio.Seq import Seq
+from Bio.Restriction import RestrictionBatch, Analysis
+import os, sys, csv
+import re
+import collections
+import copy
+import matplotlib.pyplot as plt
+import numpy as np
+import pandas as pd
+from hicstuff_log import logger
+import hicstuff_io as hio
+
+DEFAULT_FRAGMENTS_LIST_FILE_NAME = "fragments_list.txt"
+DEFAULT_INFO_CONTIGS_FILE_NAME = "info_contigs.txt"
+DEFAULT_SPARSE_MATRIX_FILE_NAME = "abs_fragments_contacts_weighted.txt"
+DEFAULT_KB_BINNING = 1
+DEFAULT_THRESHOLD_SIZE = 0
+# Most used enzyme for eukaryotes
+DEFAULT_ENZYME = "DpnII"
+# If using evenly-sized chunks instead of restriction
+# enzymes, they shouldn't be too short
+DEFAULT_MIN_CHUNK_SIZE = 50
+
+
+def write_frag_info(
+ fasta,
+ enzyme,
+ min_size=DEFAULT_THRESHOLD_SIZE,
+ circular=False,
+ output_contigs=DEFAULT_INFO_CONTIGS_FILE_NAME,
+ output_frags=DEFAULT_FRAGMENTS_LIST_FILE_NAME,
+ output_dir=None,
+):
+ """Digest and write fragment information
+ Write the fragments_list.txt and info_contigs.txt that are necessary for
+ instagraal to run.
+ Parameters
+ ----------
+ fasta : pathlib.Path or str
+ The path to the reference genome
+ enzyme : str, int or list of str
+ If a string, must be the name of an enzyme (e.g. DpnII) and the genome
+ will be cut at the enzyme's restriction sites. If a number, the genome
+ will be cut uniformly into chunks with length equal to that number. A
+ list of enzymes can also be specified if using multiple enzymes.
+ min_size : float, optional
+ Size below which shorter contigs are discarded. Default is 0, i.e. all
+ contigs are retained.
+ circular : bool, optional
+ Whether the genome is circular. Default is False.
+ output_contigs : str, optional
+ The name of the file with contig info. Default is info_contigs.txt
+ output_frags : str, optional
+ The name of the file with fragment info. Default is fragments_list.txt
+ output_dir : [type], optional
+ The path to the output directory, which will be created if not already
+ existing. Default is the current directory.
+ """
+
+ records = SeqIO.parse(hio.read_compressed(fasta), "fasta")
+
+ try:
+ info_contigs_path = os.path.join(output_dir, output_contigs)
+ frag_list_path = os.path.join(output_dir, output_frags)
+ except TypeError:
+ info_contigs_path = output_contigs
+ frag_list_path = output_frags
+
+ with open(info_contigs_path, "w") as info_contigs:
+
+ info_contigs.write("contig\tlength\tn_frags\tcumul_length\n")
+
+ with open(frag_list_path, "w") as fragments_list:
+
+ fragments_list.write(
+ "id\tchrom\tstart_pos" "\tend_pos\tsize\tgc_content\n"
+ )
+
+ total_frags = 0
+
+ for record in records:
+ contig_seq = record.seq
+ contig_name = record.id
+ contig_length = len(contig_seq)
+ if contig_length < int(min_size):
+ continue
+
+ sites = get_restriction_table(
+ contig_seq, enzyme, circular=circular
+ )
+ fragments = (
+ contig_seq[sites[i] : sites[i + 1]]
+ for i in range(len(sites) - 1)
+ )
+ n_frags = 0
+
+ current_id = 1
+ start_pos = 0
+ for frag in fragments:
+ frag_length = len(frag)
+ if frag_length > 0:
+ end_pos = start_pos + frag_length
+ gc_content = SeqUtils.GC(frag) / 100.0
+
+ current_fragment_line = "%s\t%s\t%s\t%s\t%s\t%s\n" % (
+ current_id,
+ contig_name,
+ start_pos,
+ end_pos,
+ frag_length,
+ gc_content,
+ )
+
+ fragments_list.write(current_fragment_line)
+
+ try:
+ assert (current_id == 1 and start_pos == 0) or (
+ current_id > 1 and start_pos > 0
+ )
+ except AssertionError:
+ logger.error((current_id, start_pos))
+ raise
+ start_pos = end_pos
+ current_id += 1
+ n_frags += 1
+
+ current_contig_line = "%s\t%s\t%s\t%s\n" % (
+ contig_name,
+ contig_length,
+ n_frags,
+ total_frags,
+ )
+ total_frags += n_frags
+ info_contigs.write(current_contig_line)
+
+
+def attribute_fragments(pairs_file, idx_pairs_file, restriction_table):
+ """
+ Writes the indexed pairs file, which has two more columns than the input
+ pairs file corresponding to the restriction fragment index of each read.
+ Note that pairs files have 1bp point positions whereas restriction table
+ has 0bp point poisitions.
+ Parameters
+ ----------
+ pairs_file: str
+ Path the the input pairs file. Consists of 7 tab-separated
+ columns: readID, chr1, pos1, chr2, pos2, strand1, strand2
+ idx_pairs_file: str
+ Path to the output indexed pairs file. Consists of 9 white space
+ separated columns: readID, chr1, pos1, chr2, pos2, strand1, strand2,
+ frag1, frag2. frag1 and frag2 are 0-based restriction fragments based
+ on whole genome.
+ restriction_table: dict
+ Dictionary with chromosome identifiers (str) as keys and list of
+ positions (int) of restriction sites as values.
+ """
+
+ # NOTE: Bottlenecks here are 1. binary search in find_frag and 2. writerow
+ # 1. could be reduced by searching groups of N frags in parallel and 2. by
+ # writing N frags simultaneously using a single call of writerows.
+
+ # Parse and update header section
+ pairs_header = hio.get_pairs_header(pairs_file)
+ header_size = len(pairs_header)
+ chrom_order = []
+ with open(idx_pairs_file, "w") as idx_pairs:
+ for line in pairs_header:
+ # Add new column names to header
+ if line.startswith("#columns"):
+ line = line.rstrip() + " frag1 frag2"
+ if line.startswith("#chromsize"):
+ chrom_order.append(line.split()[1])
+ idx_pairs.write(line + "\n")
+
+ # Get number of fragments per chrom to allow genome-based indices
+ shift_frags = {}
+ prev_frags = 0
+ for rank, chrom in enumerate(chrom_order):
+ if rank > 0:
+ # Note the "-1" because there are nfrags + 1 sites in rest table
+ prev_frags += len(restriction_table[chrom_order[rank - 1]]) - 1
+ # Idx of each chrom's frags will be shifted by n frags in previous chroms
+ shift_frags[chrom] = prev_frags
+
+ missing_contigs = set()
+ # Attribute pairs to fragments and append them to output file (after header)
+ with open(pairs_file, "r") as pairs, open(
+ idx_pairs_file, "a"
+ ) as idx_pairs:
+ # Skip header lines
+ for _ in range(header_size):
+ next(pairs)
+
+ # Define input and output fields
+ pairs_cols = [
+ "readID",
+ "chr1",
+ "pos1",
+ "chr2",
+ "pos2",
+ "strand1",
+ "strand2",
+ ]
+ idx_cols = pairs_cols + ["frag1", "frag2"]
+
+ # Use csv reader / writer to automatically parse columns into a dict
+ pairs_reader = csv.DictReader(
+ pairs, fieldnames=pairs_cols, delimiter="\t"
+ )
+ pairs_writer = csv.DictWriter(
+ idx_pairs, fieldnames=idx_cols, delimiter="\t"
+ )
+
+ for pair in pairs_reader:
+ # Get the 0-based indices of corresponding restriction fragments
+ # Deducing 1 from pair position to get it into 0bp point
+ pair["frag1"] = find_frag(
+ int(pair["pos1"]) - 1, restriction_table[pair["chr1"]]
+ )
+ pair["frag2"] = find_frag(
+ int(pair["pos2"]) - 1, restriction_table[pair["chr2"]]
+ )
+ # Shift fragment indices to make them genome-based instead of
+ # chromosome-based
+ try:
+ pair["frag1"] += shift_frags[pair["chr1"]]
+ except KeyError:
+ missing_contigs.add(pair["chr1"])
+ try:
+ pair["frag2"] += shift_frags[pair["chr2"]]
+ except KeyError:
+ missing_contigs.add(pair["chr2"])
+
+ # Write indexed pairs in the new file
+ pairs_writer.writerow(pair)
+
+ if missing_contigs:
+ logger.warning(
+ "Pairs on the following contigs were discarded as "
+ "those contigs are not listed in the paris file header. "
+ "This is normal if you filtered out small contigs: %s"
+ % " ".join(list(missing_contigs))
+ )
+
+
+def get_restriction_table(seq, enzyme, circular=False):
+ """
+ Get the restriction table for a single genomic sequence.
+ Parameters
+ ----------
+ seq : Seq object
+ A biopython Seq object representing a chromosomes or contig.
+ enzyme : int, str or list of str
+ The name of the restriction enzyme used, or a list of restriction
+ enzyme names. Can also be an integer, to digest by fixed chunk size.
+ circular : bool
+ Wether the genome is circular.
+ Returns
+ -------
+ numpy.array:
+ List of restriction fragment boundary positions for the input sequence.
+ >>> from Bio.Seq import Seq
+ >>> get_restriction_table(Seq("AAGCCGGATCGG"),"HpaII")
+ array([ 0, 4, 12])
+ >>> get_restriction_table(Seq("AA"),["HpaII", "MluCI"])
+ array([0, 2])
+ >>> get_restriction_table(Seq("AA"),"aeiou1")
+ Traceback (most recent call last):
+ ...
+ ValueError: aeiou1 is not a valid restriction enzyme.
+ >>> get_restriction_table("AA","HpaII")
+ Traceback (most recent call last):
+ ...
+ TypeError: Expected Seq or MutableSeq instance, got <class 'str'> instead
+ """
+ chrom_len = len(seq)
+ wrong_enzyme = "{} is not a valid restriction enzyme.".format(enzyme)
+ # Restriction batch containing the restriction enzyme
+ try:
+ enz = [enzyme] if isinstance(enzyme, str) else enzyme
+ cutter = RestrictionBatch(enz)
+ except (TypeError, ValueError):
+ try:
+ cutter = max(int(enzyme), DEFAULT_MIN_CHUNK_SIZE)
+ except ValueError:
+ raise ValueError(wrong_enzyme)
+
+ # Conversion from string type to restriction type
+ if isinstance(cutter, int):
+ sites = [i for i in range(0, chrom_len, cutter)]
+ if sites[-1] < chrom_len:
+ sites.append(chrom_len)
+ else:
+ # Find sites of all restriction enzymes given
+ ana = Analysis(cutter, seq, linear=not circular)
+ sites = ana.full()
+ # Gets all sites into a single flat list with 0-based index
+ sites = [site - 1 for enz in sites.values() for site in enz]
+ # Sort by position and allow first add start and end of seq
+ sites.sort()
+ sites.insert(0, 0)
+ sites.append(chrom_len)
+
+ return np.array(sites)
+
+
+def find_frag(pos, r_sites):
+ """
+ Use binary search to find the index of a chromosome restriction fragment
+ corresponding to an input genomic position.
+ Parameters
+ ----------
+ pos : int
+ Genomic position, in base pairs.
+ r_sites : list
+ List of genomic positions corresponding to restriction sites.
+ Returns
+ -------
+ int
+ The 0-based index of the restriction fragment to which the position belongs.
+ >>> find_frag(15, [0, 20, 30])
+ 0
+ >>> find_frag(15, [10, 20, 30])
+ Traceback (most recent call last):
+ ...
+ ValueError: The first position in the restriction table is not 0.
+ >>> find_frag(31, [0, 20, 30])
+ Traceback (most recent call last):
+ ...
+ ValueError: Read position is larger than last entry in restriction table.
+ """
+ if r_sites[0] != 0:
+ raise ValueError(
+ "The first position in the restriction table is not 0."
+ )
+ if pos > r_sites[-1]:
+ raise ValueError(
+ "Read position is larger than last entry in restriction table."
+ )
+ # binary search for the index of the read
+ index = max(np.searchsorted(r_sites, pos, side="right") - 1, 0)
+ # Last site = end of the chrom, index of last fragment is last site - 1
+ index = min(len(r_sites) - 2, index)
+
+ return index
+
+
+def frag_len(
+ frags_file_name=DEFAULT_FRAGMENTS_LIST_FILE_NAME,
+ output_dir=None,
+ plot=False,
+ fig_path=None,
+):
+ """
+ logs summary statistics of fragment length distribution based on an
+ input fragment file. Can optionally show a histogram instead
+ of text summary.
+ Parameters
+ ----------
+ frags_file_name : str
+ Path to the output list of fragments.
+ output_dir : str
+ Directory where the list should be saved.
+ plot : bool
+ Wether a histogram of fragment length should be shown.
+ fig_path : str
+ If a path is given, the figure will be saved instead of shown.
+ """
+
+ try:
+ frag_list_path = os.path.join(output_dir, frags_file_name)
+ except TypeError:
+ frag_list_path = frags_file_name
+ frags = pd.read_csv(frag_list_path, sep="\t")
+ nfrags = frags.shape[0]
+ med_len = frags["size"].median()
+ nbins = 40
+ if plot:
+ fig, ax = plt.subplots()
+ _, _, _ = ax.hist(frags["size"], bins=nbins)
+
+ ax.set_xlabel("Fragment length [bp]")
+ ax.set_ylabel("Log10 number of fragments")
+ ax.set_title("Distribution of restriction fragment length")
+ ax.set_yscale("log", base=10)
+ ax.annotate(
+ "Total fragments: {}".format(nfrags),
+ xy=(0.95, 0.95),
+ xycoords="axes fraction",
+ fontsize=12,
+ horizontalalignment="right",
+ verticalalignment="top",
+ )
+ ax.annotate(
+ "Median length: {}".format(med_len),
+ xy=(0.95, 0.90),
+ xycoords="axes fraction",
+ fontsize=12,
+ horizontalalignment="right",
+ verticalalignment="top",
+ )
+ # Tweak spacing to prevent clipping of ylabel
+ fig.tight_layout()
+ if fig_path:
+ plt.savefig(fig_path)
+ else:
+ plt.show()
+ plt.clf()
+ else:
+ logger.info(
+ "Genome digested into {0} fragments with a median "
+ "length of {1}".format(nfrags, med_len)
+ )
+
+
+def gen_enzyme_religation_regex(enzyme):
+ """Return a regex which corresponds to all possible religation sites given a
+ set of enzyme.
+ Parameters:
+ -----------
+ enzyme : str
+ String that contains the names of the enzyme separated by a comma.
+ Returns:
+ --------
+ re.Pattern :
+ Regex that corresponds to all possible ligation sites given a set of
+ enzyme.
+ Examples:
+ ---------
+ >>> gen_enzyme_religation_regex('HpaII')
+ re.compile('CCGCGG')
+ >>> gen_enzyme_religation_regex('HpaII,MluCI')
+ re.compile('AATTAATT|AATTCGG|CCGAATT|CCGCGG')
+ """
+
+ # Split the str on the comma to separate the different enzymes.
+ enzyme = enzyme.split(",")
+
+ # Check on Biopython dictionnary the enzyme.
+ rb = RestrictionBatch(enzyme)
+
+ # Initiation:
+ give_list = []
+ accept_list = []
+ ligation_list = []
+
+ # Iterates on the enzymes.
+ for enz in rb:
+
+ # Extract restriction sites and look for cut sites.
+ site = enz.elucidate()
+ fw_cut = site.find("^")
+ rev_cut = site.find("_")
+
+ # Process "give" site. Remove N on the left (useless).
+ give_site = site[:rev_cut].replace("^", "")
+ while give_site[0] == "N":
+ give_site = give_site[1:]
+ give_list.append(give_site)
+
+ # Process "accept" site. Remove N on the rigth (useless).
+ accept_site = site[fw_cut + 1 :].replace("_", "")
+ while accept_site[-1] == "N":
+ accept_site = accept_site[:-1]
+ accept_list.append(accept_site)
+
+ # Iterates on the two list to build all the possible HiC ligation sites.
+ for give_site in give_list:
+ for accept_site in accept_list:
+ # Replace "N" by "." for regex searching of the sites
+ ligation_list.append((give_site + accept_site).replace("N", "."))
+ ligation_list.append(
+ str(Seq(give_site + accept_site).reverse_complement()).replace(
+ "N", "."
+ )
+ )
+
+ # Build the regex for any ligation sites.
+ pattern = "|".join(sorted(list(set(ligation_list))))
+ return re.compile(pattern)
diff --git a/bin/hicstuff_fragments.py b/bin/hicstuff_fragments.py
new file mode 100755
index 0000000..c49378e
--- /dev/null
+++ b/bin/hicstuff_fragments.py
@@ -0,0 +1,40 @@
+#!/usr/bin/env python
+
+import hicstuff_digest as hcd
+import argparse
+
+if __name__ == "__main__":
+ parser = argparse.ArgumentParser()
+ parser.add_argument("-e", "--enzyme")
+ parser.add_argument("-i", "--index")
+ args = parser.parse_args()
+
+ enzyme = args.enzyme
+ index = args.index
+
+
+ #hicstuff case sensitive enzymes adaptation
+ if enzyme == "hindiii":
+ enzyme = "HindIII"
+ elif enzyme == "dpnii":
+ enzyme = "DpnII"
+ elif enzyme == "bglii":
+ enzyme = "BglII"
+ elif enzyme == "mboi":
+ enzyme = "MboI"
+ elif enzyme == "arima":
+ enzyme = "Arima"
+
+
+ # Generate info_contigs and fragments_list output files
+ hcd.write_frag_info(
+ index,
+ enzyme,
+ min_size=0,
+ circular=False,
+ output_contigs="info_contigs.txt",
+ output_frags="fragments_list.txt",
+ )
+
+ # Log fragment size distribution
+ hcd.frag_len(frags_file_name="fragments_list.txt", plot=False, fig_path="frags_hist.pdf")
diff --git a/bin/hicstuff_io.py b/bin/hicstuff_io.py
new file mode 100644
index 0000000..c8c4dde
--- /dev/null
+++ b/bin/hicstuff_io.py
@@ -0,0 +1,1379 @@
+#!/usr/bin/env python3
+# -*- coding: utf-8 -*-
+
+import gzip
+import zipfile
+import bz2
+import io
+import os
+import functools
+import sys
+import numpy as np
+import pandas as pd
+import collections
+import subprocess as sp
+from scipy.sparse import tril, triu
+import pathlib
+import re
+from os.path import join, exists
+from random import getrandbits
+from scipy.sparse import coo_matrix
+from Bio import SeqIO, SeqUtils
+#import hicstuff.hicstuff as hcs
+#from hicstuff.log import logger
+#from hicstuff.version import __version__
+import scipy.stats as ss
+
+DEFAULT_MAX_MATRIX_SHAPE = 10000
+
+DEFAULT_FRAGMENTS_LIST_FILE_NAME = "fragments_list.txt"
+DEFAULT_INFO_CONTIGS_FILE_NAME = "info_contigs.txt"
+DEFAULT_SPARSE_MATRIX_FILE_NAME = "abs_fragments_contacts_weighted.txt"
+
+
+def _cols_to_sparse(sparse_array, shape=None, dtype=np.float64):
+ """
+ Make a coordinate based sparse matrix from columns.
+ Convert (3, n) shaped arrays to a sparse matrix. The first
+ one acts as the row coordinates, the second one as the
+ column coordinates, the third one as the data points
+ for each pair. If duplicates are found, the data points are
+ added.
+ Parameters
+ ----------
+ sparse_array : array_like
+ An array with exactly three columns representing the sparse
+ matrix data in coordinate format.
+ shape : tuple of int
+ The total number of rows and columns in the matrix. Will be estimated
+ from nonzero values if omitted.
+ dtype : type, optional
+ The type of data being loaded. Default is numpy.float64
+ Example
+ -------
+ >>> import numpy as np
+ >>> row, col = np.array([1, 2, 3]), np.array([3, 2, 1])
+ >>> data = np.array([4, 5, 6])
+ >>> M = np.array([row, col, data]).T
+ >>> S = _cols_to_sparse(M)
+ >>> print(S.todense())
+ [[0. 0. 0. 0.]
+ [0. 0. 0. 4.]
+ [0. 0. 5. 0.]
+ [0. 6. 0. 0.]]
+ """
+
+ row = sparse_array[:, 0]
+ col = sparse_array[:, 1]
+ data = sparse_array[:, 2]
+ if shape is None:
+ n = int(max(np.amax(row), np.amax(col))) + 1
+ shape = (n, n)
+
+ S = coo_matrix((data, (row, col)), shape=shape, dtype=dtype)
+ return S
+
+
+def load_sparse_matrix(mat_path, binning=1, dtype=np.float64):
+ """Load sparse matrix
+ Load a text file matrix into a sparse matrix object. The expected format is
+ a 3 column file where columns are row_number, col_number, value. The first
+ line consists of 3 values representing the total number of rows, columns
+ and nonzero values.
+ Parameters
+ ----------
+ mat_path : file, str or pathlib.Path
+ The input matrix file in instagraal format.
+ binning : int or "auto"
+ The binning to perform. If "auto", binning will
+ be automatically inferred so that the matrix size
+ will not go beyond (10000, 10000) in shape. That
+ can be changed by modifying the DEFAULT_MAX_MATRIX_SHAPE
+ value. Default is 1, i.e. no binning is performed
+ dtype : type, optional
+ The type of data being loaded. Default is numpy.float64
+ Returns
+ -------
+ sparse_mat : scipy.sparse.coo_matrix
+ The output (sparse) matrix in COOrdinate format.
+ Examples
+ --------
+ >>> S = load_sparse_matrix('test_data/mat_5kb.tsv', binning=1)
+ >>> S.data[:10]
+ array([84., 2., 3., 2., 1., 1., 50., 1., 66., 1.])
+ >>> S.shape
+ (16, 16)
+ """
+ try:
+ cols_arr = np.loadtxt(mat_path, delimiter="\t", dtype=dtype)
+ shape = (int(cols_arr[0, 0]), int(cols_arr[0, 1]))
+ except ValueError:
+ cols_arr = np.loadtxt(
+ mat_path, delimiter="\t", dtype=dtype, skiprows=1
+ )
+ shape = None
+
+ # Get values into an array without the header. Use the header to give size.
+ sparse_mat = _cols_to_sparse(cols_arr[1:, :], shape=shape, dtype=dtype)
+
+ if binning == "auto":
+ num_bins = max(sparse_mat.shape) + 1
+ subsampling_factor = num_bins // DEFAULT_MAX_MATRIX_SHAPE
+ else:
+ subsampling_factor = binning
+ sparse_mat = hcs.bin_sparse(
+ sparse_mat, subsampling_factor=subsampling_factor
+ )
+ return sparse_mat
+
+
+def save_sparse_matrix(s_mat, path):
+ """Save a sparse matrix
+ Saves a sparse matrix object into tsv format.
+ Parameters
+ ----------
+ s_mat : scipy.sparse.coo_matrix
+ The sparse matrix to save on disk
+ path : str
+ File path where the matrix will be stored
+ """
+ if s_mat.format != "coo":
+ ValueError("Sparse matrix must be in coo format")
+ dtype = s_mat.dtype
+ fmt = "%i" if dtype == int else "%.10e"
+ sparse_arr = np.vstack([s_mat.row, s_mat.col, s_mat.data]).T
+
+ np.savetxt(
+ path,
+ sparse_arr,
+ header="{nrows}\t{ncols}\t{nonzero}".format(
+ nrows=s_mat.shape[0], ncols=s_mat.shape[1], nonzero=s_mat.nnz
+ ),
+ comments="",
+ fmt=fmt,
+ delimiter="\t",
+ )
+
+
+def load_pos_col(path, colnum, header=1, dtype=np.int64):
+ """
+ Loads a single column of a TSV file with header into a numpy array.
+
+ Parameters
+ ----------
+ path : str
+ The path of the TSV file to load.
+ colnum : int
+ The 0-based index of the column to load.
+ header : int
+ Number of line to skip. By default the header is a single line.
+ Returns
+ -------
+ numpy.array :
+ A 1D numpy array with the
+ Examples
+ --------
+ >>> load_pos_col('test_data/mat_5kb.tsv', 0)[:10]
+ array([0, 0, 0, 0, 0, 0, 1, 1, 2, 2])
+ """
+ pos_arr = np.genfromtxt(
+ path,
+ delimiter="\t",
+ usecols=(colnum,),
+ skip_header=header,
+ dtype=dtype,
+ )
+ return pos_arr
+
+
+def generate_temp_dir(path):
+ """Temporary directory generation
+ Generates a temporary file with a random name at the input path.
+
+ Parameters
+ ----------
+ path : str
+ The path at which the temporary directory will be created.
+
+ Returns
+ -------
+ str
+ The path of the newly created temporary directory.
+ """
+ exist = True
+ while exist:
+ # Keep trying random directory names if they already exist
+ directory = str(hex(getrandbits(32)))[2:]
+ full_path = join(path, directory)
+ if not exists(full_path):
+ exist = False
+ try:
+ os.makedirs(full_path)
+ except PermissionError:
+ raise PermissionError(
+ "The temporary directory cannot be created in {}. "
+ "Make sure you have write permission.".format(path)
+ )
+ return full_path
+
+
+def _check_cooler(fun):
+ """Decorates function `fun` to check if cooler is available.."""
+
+ @functools.wraps(fun)
+ def wrapped(*args, **kwargs):
+ try:
+ import cooler
+
+ fun.__globals__["cooler"] = cooler
+ except ImportError:
+ logger.error(
+ "The cooler package is required to use {0}, please install it first".format(
+ fun.__name__
+ )
+ )
+ raise ImportError("The cooler package is required.")
+ return fun(*args, **kwargs)
+
+ return wrapped
+
+
+@_check_cooler
+def add_cool_column(
+ clr, column, column_name, table_name="bins", metadata={}, dtype=None
+):
+ """
+ Adds a new column to a loaded Cooler store. If the column exists,
+ it is replaced. This will affect the .cool file.
+
+ Parameters
+ ----------
+ clr : Cooler object
+ A Cooler store.
+ column : pandas Series
+ The column to add to the cooler.
+ column_name : str
+ The name of the column to add.
+ table_name : str
+ The name of the table to which the column should be added.
+ Defaults to the "bins" table.
+ metadata : dict
+ A dictionary of metadata to associate with the new column.
+ """
+ with clr.open("r+") as c:
+ if column_name in c[table_name]:
+ del c[table_name][column_name]
+ h5opts = dict(compression="gzip", compression_opts=6)
+ c[table_name].create_dataset(
+ column_name, data=column, dtype=dtype, **h5opts
+ )
+ c[table_name][column_name].attrs.update(metadata)
+
+
+@_check_cooler
+def load_cool(cool):
+ """
+ Reads a cool file into memory and parses it into graal style tables.
+
+ Parameters
+ ----------
+ cool : str
+ Path to the input .cool file.
+ Returns
+ -------
+ mat : scipy coo_matrix
+ Hi-C contact map in COO format.
+ frags : pandas DataFrame
+ Table off bins matching the matrix. Corresponds to the content of the fragments_list.txt file.
+ chroms : pandas DataFrame
+ Table of chromosome informations.
+ """
+ c = cooler.Cooler(cool) # pylint: disable=undefined-variable
+ frags = c.bins()[:]
+ chroms = c.chroms()[:]
+ mat = c.pixels()[:]
+ frags.rename(
+ columns={"start": "start_pos", "end": "end_pos"}, inplace=True
+ )
+ frags["id"] = frags.groupby("chrom", sort=False).cumcount() + 1
+ # Try loading hicstuff-specific columns
+ try:
+ frags = frags[
+ ["id", "chrom", "start_pos", "end_pos", "size", "gc_content"]
+ ]
+ # If absent, only load standard columns
+ except KeyError:
+ frags = frags[["id", "chrom", "start_pos", "end_pos"]]
+
+ chroms["cumul_length"] = (
+ chroms.length.shift(1).fillna(0).cumsum().astype(int)
+ )
+ n_frags = c.bins()[:].groupby("chrom", sort=False).count().start
+ chroms["n_frags"] = chroms.merge(
+ n_frags, right_index=True, left_on="name", how="left"
+ ).start
+ chroms.rename(columns={"name": "contig"}, inplace=True)
+ n = int(max(np.amax(mat.bin1_id), np.amax(mat.bin2_id))) + 1
+ shape = (n, n)
+ mat = coo_matrix((mat["count"], (mat.bin1_id, mat.bin2_id)), shape=shape)
+
+ return mat, frags, chroms
+
+
+@_check_cooler
+def save_cool(cool_out, mat, frags, metadata={}):
+ """
+ Writes a .cool file from graal style tables.
+
+ Parameters
+ ----------
+ cool_out : str
+ Path to the output cool file.
+ mat : scipy coo_matrix
+ The Hi-C contact matrix in sparse COO format.
+ frags : pandas DataFrame
+ The graal style 'fragments_list' table.
+ metadata : dict
+ Potential metadata to associate with the cool file.
+ """
+ up_tri = False
+ # Check if symmetric matrix is symmetric
+ # (i.e. only upper triangle or full mat)
+ if (abs(mat - mat.T) > 1e-10).nnz != 0:
+ up_tri = True
+ # Drop useless column
+ try:
+ bins = frags.drop("id", axis=1)
+ except KeyError:
+ bins = frags
+ # Get column names right
+ bins.rename(
+ columns={"seq": "chrom", "start_pos": "start", "end_pos": "end"},
+ inplace=True,
+ )
+ mat_dict = {"bin1_id": mat.row, "bin2_id": mat.col, "count": mat.data}
+ pixels = pd.DataFrame(mat_dict)
+ cooler.create_cooler( # pylint: disable=undefined-variable
+ cool_out,
+ bins,
+ pixels,
+ metadata=metadata,
+ symmetric_upper=up_tri,
+ triucheck=False,
+ )
+
+
+def read_compressed(filename, mode='r'):
+ """Read compressed file
+ Opens the file in read mode with appropriate decompression algorithm.
+ Parameters
+ ----------
+ filename : str
+ The path to the input file
+ Returns
+ -------
+ file-like object
+ The handle to access the input file's content
+ """
+
+ # Standard header bytes for diff compression formats
+ comp_bytes = {
+ b"\x1f\x8b\x08": "gz",
+ b"\x42\x5a\x68": "bz2",
+ b"\x50\x4b\x03\x04": "zip",
+ }
+
+ max_len = max(len(x) for x in comp_bytes)
+
+ def file_type(filename):
+ """Guess file type
+ Compare header bytes with those in the file and return type.
+ """
+ with open(filename, "rb") as f:
+ file_start = f.read(max_len)
+ for magic, filetype in comp_bytes.items():
+ if file_start.startswith(magic):
+ return filetype
+ return "uncompressed"
+
+ # Open file with appropriate function
+ mode_map = {'r': 'rt', 'rb': 'rb'}
+ comp = file_type(filename)
+ if comp == "gz":
+ return gzip.open(filename, mode_map[mode])
+ elif comp == "bz2":
+ return bz2.open(filename, mode_map[mode])
+ elif comp == "zip":
+ zip_arch = zipfile.ZipFile(filename, mode)
+ if len(zip_arch.namelist()) > 1:
+ raise IOError(
+ "Only a single fastq file must be in the zip archive."
+ )
+ else:
+ # ZipFile opens as bytes by default, using io to read as text
+ zip_content = zip_arch.open(zip_arch.namelist()[0], mode)
+ return io.TextIOWrapper(zip_content, encoding="utf-8")
+ else:
+ return open(filename, mode)
+
+
+def is_compressed(filename):
+ """Check compression status
+ Check if the input file is compressed from the first bytes.
+ Parameters
+ ----------
+ filename : str
+ The path to the input file
+ Returns
+ -------
+ bool
+ True if the file is compressed, False otherwise.
+ """
+
+ # Standard header bytes for diff compression formats
+ comp_bytes = {
+ b"\x1f\x8b\x08": "gz",
+ b"\x42\x5a\x68": "bz2",
+ b"\x50\x4b\x03\x04": "zip",
+ }
+ max_len = max(len(x) for x in comp_bytes)
+ with open(filename, "rb") as f:
+ file_start = f.read(max_len)
+ for magic, _ in comp_bytes.items():
+ if file_start.startswith(magic):
+ return True
+ return False
+
+
+def from_dade_matrix(filename, header=False):
+ """Load a DADE matrix
+ Load a numpy array from a DADE matrix file, optionally
+ returning bin information from the header. Header data
+ processing is delegated downstream.
+ Parameters
+ ----------
+ filename : str, file or pathlib.Path
+ The name of the file containing the DADE matrix.
+ header : bool
+ Whether to return as well information contained
+ in the header. Default is False.
+ Example
+ -------
+ >>> import numpy as np
+ >>> import tempfile
+ >>> lines = [['RST', 'chr1~0', 'chr1~10', 'chr2~0', 'chr2~30'],
+ ... ['chr1~0', '5'],
+ ... ['chr1~10', '8', '3'],
+ ... ['chr2~0', '3', '5', '5'],
+ ... ['chr2~30', '5', '10', '11', '2']
+ ... ]
+ >>> formatted = ["\\t".join(l) + "\\n" for l in lines ]
+ >>> dade = tempfile.NamedTemporaryFile(mode='w')
+ >>> for fm in formatted:
+ ... dade.write(fm)
+ 34
+ 9
+ 12
+ 13
+ 18
+ >>> dade.flush()
+ >>> M, h = from_dade_matrix(dade.name, header=True)
+ >>> dade.close()
+ >>> print(M)
+ [[ 5. 8. 3. 5.]
+ [ 8. 3. 5. 10.]
+ [ 3. 5. 5. 11.]
+ [ 5. 10. 11. 2.]]
+ >>> print(h)
+ ['chr1~0', 'chr1~10', 'chr2~0', 'chr2~30']
+ See https://github.com/scovit/DADE for more details about Dade.
+ """
+
+ A = pd.read_csv(filename, sep="\t", header=None)
+ A.fillna("0", inplace=True)
+ M, headers = np.array(A.iloc[1:, 1:], dtype=np.float64), A.iloc[0, :]
+ matrix = M + M.T - np.diag(np.diag(M))
+ if header:
+ return matrix, headers.tolist()[1:]
+ else:
+ return matrix
+
+
+def to_dade_matrix(M, annotations="", filename=None):
+ """Returns a Dade matrix from input numpy matrix. Any annotations are added
+ as header. If filename is provided and valid, said matrix is also saved
+ as text.
+ """
+
+ n, m = M.shape
+ A = np.zeros((n + 1, m + 1))
+ A[1:, 1:] = M
+ if not annotations:
+ annotations = np.array(["" for _ in n], dtype=str)
+ A[0, :] = annotations
+ A[:, 0] = annotations.T
+ if filename:
+ try:
+ np.savetxt(filename, A, fmt="%i")
+ logger.info(
+ "I saved input matrix in dade format as {0}".format(
+ str(filename)
+ )
+ )
+ except ValueError as e:
+ logger.warning("I couldn't save input matrix.")
+ logger.warning(str(e))
+
+ return A
+
+
+def load_into_redis(filename):
+ """Load a file into redis
+ Load a matrix file and sotre it in memory with redis.
+ Useful to pass around huge datasets from scripts to
+ scripts and load them only once.
+ Inspired from https://gist.github.com/alexland/ce02d6ae5c8b63413843
+ Parameters
+ ----------
+ filename : str, file or pathlib.Path
+ The file of the matrix to load.
+ Returns
+ -------
+ key : str
+ The key of the dataset needed to retrieve it from redis.
+ """
+ try:
+ from redis import StrictRedis as redis
+ import time
+ except ImportError:
+ print(
+ "Error! Redis does not appear to be installed in your system.",
+ file=sys.stderr,
+ )
+ exit(1)
+
+ M = np.genfromtxt(filename, dtype=None)
+ array_dtype = str(M.dtype)
+ m, n = M.shape
+ M = M.ravel().tostring()
+ database = redis(host="localhost", port=6379, db=0)
+ key = "{0}|{1}#{2}#{3}".format(int(time.time()), array_dtype, m, n)
+
+ database.set(key, M)
+
+ return key
+
+
+def load_from_redis(key):
+ """Retrieve a dataset from redis
+ Retrieve a cached dataset that was stored in redis
+ with the input key.
+ Parameters
+ ----------
+ key : str
+ The key of the dataset that was stored in redis.
+ Returns
+ -------
+ M : numpy.ndarray
+ The retrieved dataset in array format.
+ """
+
+ try:
+ from redis import StrictRedis as redis
+ except ImportError:
+ print(
+ "Error! Redis does not appear to be installed in your system.",
+ file=sys.stderr,
+ )
+ exit(1)
+
+ database = redis(host="localhost", port=6379, db=0)
+
+ try:
+ M = database.get(key)
+ except KeyError:
+ print(
+ "Error! No dataset was found with the supplied key.",
+ file=sys.stderr,
+ )
+ exit(1)
+
+ array_dtype, n, m = key.split("|")[1].split("#")
+
+ M = np.fromstring(M, dtype=array_dtype).reshape(int(n), int(m))
+ return M
+
+
+def dade_to_graal(
+ filename,
+ output_matrix=DEFAULT_SPARSE_MATRIX_FILE_NAME,
+ output_contigs=DEFAULT_INFO_CONTIGS_FILE_NAME,
+ output_frags=DEFAULT_SPARSE_MATRIX_FILE_NAME,
+ output_dir=None,
+):
+ """Convert a matrix from DADE format (https://github.com/scovit/dade)
+ to a graal-compatible format. Since DADE matrices contain both fragment
+ and contact information all files are generated at the same time.
+ """
+
+ with open(output_matrix, "w") as sparse_file:
+ sparse_file.write("id_frag_a\tid_frag_b\tn_contact")
+ with open(filename) as file_handle:
+ first_line = file_handle.readline()
+ for row_index, line in enumerate(file_handle):
+ dense_row = np.array(line.split("\t")[1:], dtype=np.int32)
+ for col_index in np.nonzero(dense_row)[0]:
+ line_to_write = "{}\t{}\t{}\n".format(
+ row_index, col_index, dense_row[col_index]
+ )
+ sparse_file.write(line_to_write)
+
+ header = first_line.split("\t")
+ bin_type = header[0]
+ if bin_type == '"RST"':
+ logger.info("I detected fragment-wise binning")
+ elif bin_type == '"BIN"':
+ logger.info("I detected fixed size binning")
+ else:
+ logger.warning(
+ (
+ "Sorry, I don't understand this matrix's "
+ "binning: I read {}".format(str(bin_type))
+ )
+ )
+
+ header_data = [
+ header_elt.replace("'", "")
+ .replace('"', "")
+ .replace("\n", "")
+ .split("~")
+ for header_elt in header[1:]
+ ]
+
+ (
+ global_frag_ids,
+ contig_names,
+ local_frag_ids,
+ frag_starts,
+ frag_ends,
+ ) = np.array(list(zip(*header_data)))
+
+ frag_starts = frag_starts.astype(np.int32) - 1
+ frag_ends = frag_ends.astype(np.int32) - 1
+ frag_lengths = frag_ends - frag_starts
+
+ total_length = len(global_frag_ids)
+
+ with open(output_contigs, "w") as info_contigs:
+
+ info_contigs.write("contig\tlength\tn_frags\tcumul_length\n")
+
+ cumul_length = 0
+
+ for contig in collections.OrderedDict.fromkeys(contig_names):
+
+ length_tig = np.sum(frag_lengths[contig_names == contig])
+ n_frags = collections.Counter(contig_names)[contig]
+ line_to_write = "%s\t%s\t%s\t%s\n" % (
+ contig,
+ length_tig,
+ n_frags,
+ cumul_length,
+ )
+ info_contigs.write(line_to_write)
+ cumul_length += n_frags
+
+ with open(output_frags, "w") as fragments_list:
+
+ fragments_list.write(
+ "id\tchrom\tstart_pos\tend_pos" "\tsize\tgc_content\n"
+ )
+ bogus_gc = 0.5
+
+ for i in range(total_length):
+ line_to_write = "%s\t%s\t%s\t%s\t%s\t%s\n" % (
+ int(local_frag_ids[i]) + 1,
+ contig_names[i],
+ frag_starts[i],
+ frag_ends[i],
+ frag_lengths[i],
+ bogus_gc,
+ )
+ fragments_list.write(line_to_write)
+
+
+def load_bedgraph2d(filename, bin_size=None, fragments_file=None):
+ """
+ Loads matrix and fragment information from a 2D bedgraph file. Note this
+ function assumes chromosomes are ordered in alphabetical. order
+
+ Parameters
+ ----------
+ filename : str
+ Path to the bedgraph2D file.
+ bin_size : int
+ The size of bins in the case of fixed bin size.
+ fragments_file : str
+ Path to a fragments file to explicitely provide fragments positions.
+ If the matrix does not have fixed bin size, this prevents errors.
+
+ Returns
+ -------
+ mat : scipy.sparse.coo_matrix
+ The Hi-C contact map as the upper triangle of a symetric matrix, in
+ sparse format.
+ frags : pandas.DataFrame
+ The list of fragments/bin present in the matrix with their genomic
+ positions.
+ """
+ bed2d = pd.read_csv(filename, sep="\t", header=None)
+ chrom_sizes = {}
+ if bin_size is not None:
+ # If bin size if provided, retrieve chromosome lengths, this will be
+ # used when regenerating bin coordinates
+ chroms_left = bed2d[[3, 5]]
+ chroms_left.columns = [0, 2]
+ chroms = (
+ pd.concat([bed2d[[0, 2]], chroms_left])
+ .groupby([0], sort=False)
+ .max()
+ )
+ for chrom, size in zip(chroms.index, np.array(chroms)):
+ chrom_sizes[chrom] = size[0]
+ elif fragments_file is None:
+ logger.warning(
+ "Please be aware that not all information can be restored from a "
+ "bg2 file without fixed bin size; fragments without any contact "
+ "will be lost"
+ )
+ # Get all possible fragment chrom-positions into an array
+ frag_pos = np.vstack(
+ [np.array(bed2d[[0, 1, 2]]), np.array(bed2d[[3, 4, 5]])]
+ )
+ # Sort by position (least important, col 1)
+ frag_pos = frag_pos[frag_pos[:, 1].argsort(kind="mergesort")]
+ # Then by chrom (most important, col 0)
+ frag_pos = frag_pos[frag_pos[:, 0].argsort(kind="mergesort")]
+ # Get unique names for fragments (chrom+pos)
+ ordered_frag_pos = (
+ pd.DataFrame(frag_pos).drop_duplicates().reset_index(drop=True)
+ )
+ frag_pos_a = bed2d[[0, 1]].apply(lambda x: tuple(x), axis=1)
+ frag_pos_b = bed2d[[3, 4]].apply(lambda x: tuple(x), axis=1)
+ # If fragments file is provided, use fragments positions to indices mapping
+ if fragments_file is not None:
+ frags = pd.read_csv(fragments_file, delimiter="\t")
+ frag_map = frags.apply(lambda x: (str(x.chrom), x.start_pos), axis=1)
+ frag_map = {f_name: f_idx for f_idx, f_name in enumerate(frag_map)}
+ # If fixed fragment size available, use it to reconstruct original
+ # fragments ID (even if they are absent from the bedgraph file).
+ elif bin_size is not None:
+ frag_map = {}
+ chrom_frags = []
+ for chrom, size in chrom_sizes.items():
+ prev_frags = len(frag_map)
+ for bin_id, bin_pos in enumerate(range(0, size, bin_size)):
+ frag_map[(chrom, bin_pos)] = bin_id + prev_frags
+ n_bins = size // bin_size
+ chrom_frags.append(
+ pd.DataFrame(
+ {
+ "id": range(1, n_bins + 1),
+ "chrom": np.repeat(chrom, n_bins),
+ "start_pos": range(0, size, bin_size),
+ "end_pos": range(bin_size, size + bin_size, bin_size),
+ }
+ )
+ )
+ frags = pd.concat(chrom_frags, axis=0).reset_index(drop=True)
+ frags.insert(
+ loc=3, column="size", value=frags.end_pos - frags.start_pos
+ )
+ # If None available, guess fragments indices from bedgraph (potentially wrong)
+ else:
+ frag_map = {
+ (v[0], v[1]): i
+ for i, v in ordered_frag_pos.iloc[:, [0, 1]].iterrows()
+ }
+ frags = ordered_frag_pos.copy()
+ frags[3] = frags.iloc[:, 2] - frags.iloc[:, 1]
+ frags.insert(loc=0, column="id", value=0)
+ frags.id = frags.groupby([0], sort=False).cumcount() + 1
+ frags.columns = ["id", "chrom", "start_pos", "end_pos", "size"]
+ # Match bin indices to their names
+ frag_id_a = np.array(list(map(lambda x: frag_map[x], frag_pos_a)))
+ frag_id_b = np.array(list(map(lambda x: frag_map[x], frag_pos_b)))
+ contacts = np.array(bed2d.iloc[:, 6].tolist())
+ # Use index to build matrix
+ n_frags = len(frag_map.keys())
+ mat = coo_matrix(
+ (contacts, (frag_id_a, frag_id_b)), shape=(n_frags, n_frags)
+ )
+
+ # Get size of each chromosome in basepairs
+ chromsizes = frags.groupby("chrom", sort=False).apply(
+ lambda x: np.int64(max(x.end_pos))
+ )
+ chrom_bins = frags.groupby("chrom", sort=False).size()
+ # Shift chromsizes by one to get starting bin, first one is zero
+ # Make chromsize cumulative to get start bin of each chrom
+ # Get chroms into a 1D array of bin starts
+ chrom_start = chrom_bins.shift(1, fill_value=0).cumsum()
+ chroms = pd.DataFrame(
+ {
+ "contig": chromsizes.index,
+ "length": chromsizes.values,
+ "n_frags": chrom_bins,
+ "cumul_length": chrom_start,
+ }
+ )
+ return mat, frags, chroms
+
+
+def flexible_hic_loader(
+ mat, fragments_file=None, chroms_file=None, quiet=False
+):
+ """
+ Wrapper function to load COO, bg2 or cool input and return the same output.
+ COO formats requires fragments_file and chroms_file options. bg2 format can
+ infer bin_size if fixed. When providing a bg2 matrix with uneven fragments
+ length, one should provide fragments_file as well or empty bins will be
+ truncated from the output.
+
+ Parameters
+ ----------
+ mat : str
+ Path to the matrix in graal, bedgraph2 or cool format.
+ fragments_file : str or None
+ Path to the file with fragments information (fragments_list.txt).
+ Only required if the matrix is in graal format.
+ chroms_file : str or None
+ Path to the file with chromosome information (info_contigs.txt). Only required
+ if the matrix is in graal format.
+ quiet : bool
+ If True, will silence warnings for empty outputs.
+ Returns
+ -------
+ mat : scipy.sparse.coo_matrix
+ Sparse upper triangle Hi-C matrix.
+ frags : pandas.DataFrame or None
+ Table of fragment informations. None if information was not provided.
+ chroms : pandas.DataFrame or None
+ Table of chromosomes/contig information. None if information was not provided.
+ """
+ hic_format = get_hic_format(mat)
+ # Load cool based on file extension
+ if hic_format == "cool":
+ mat, frags, chroms = load_cool(mat)
+ # Use the first line to determine COO / bg2 format
+ if hic_format == "bg2":
+ # Use the frags file to define bins if available
+ if fragments_file is not None:
+ mat, frags, chroms = load_bedgraph2d(
+ mat, fragments_file=fragments_file
+ )
+ else:
+ # Guess if bin size is fixed based on MAD
+ bg2 = pd.read_csv(mat, sep="\t")
+ sizes = np.array(bg2.iloc[:, 2] - bg2.iloc[:, 1])
+ size_mad = ss.median_abs_deviation(sizes, scale='normal')
+ # Use only the bg2
+ if size_mad > 0:
+ mat, frags, chroms = load_bedgraph2d(mat)
+ logger.warning(
+ "Input is a bedgraph2d file with uneven bin size, "
+ "but no fragments_file was provided. Empty bins will "
+ "be missing from the output. To avoid this, provide a "
+ "fragments file."
+ )
+ # Use fixed bin size
+ else:
+ mat, frags, chroms = load_bedgraph2d(
+ mat, bin_size=int(np.median(sizes))
+ )
+
+ elif hic_format == "graal":
+ mat = load_sparse_matrix(mat)
+ try:
+ frags = pd.read_csv(fragments_file, sep="\t")
+ except ValueError:
+ if not quiet:
+ logger.warning(
+ "fragments_file was not provided when "
+ "loading a matrix in COO/graal format. frags will be None."
+ )
+ frags = None
+ try:
+ chroms = pd.read_csv(chroms_file, sep="\t")
+ except ValueError:
+ if not quiet:
+ logger.warning(
+ "chroms_file was not provided when "
+ "loading a matrix in COO/graal format. chroms will be None."
+ )
+
+ chroms = None
+
+ # Ensure the matrix is upper triangle symmetric
+ if mat.shape[0] == mat.shape[1]:
+ if (abs(mat - mat.T) > 1e-10).nnz > 0:
+ mat = mat + tril(mat, k=-1).T
+ mat = triu(mat, format="coo")
+
+ return mat, frags, chroms
+
+
+def get_hic_format(mat):
+ """Returns the format of the input Hi-C matrix
+
+ Parameters
+ ----------
+ mat : str
+ Path to the input Hi-C matrix
+ Returns
+ -------
+ str :
+ Hi-C format string. One of graal, bg2, cool
+ """
+ if (
+ mat.endswith(".cool")
+ or mat.count(".mcool::/") == 1
+ or mat.count(".cool::/") == 1
+ ):
+ hic_format = "cool"
+ else:
+ # Use the first line to determine COO / bg2 format
+ ncols = len(open(mat).readline().split("\t"))
+ if ncols == 7:
+ hic_format = "bg2"
+ elif ncols == 3:
+ hic_format = "graal"
+ else:
+ raise ValueError("Unkown file format")
+ return hic_format
+
+
+def flexible_hic_saver(
+ mat, out_prefix, frags=None, chroms=None, hic_fmt="graal", quiet=False,
+):
+ """
+ Saves objects to the desired Hi-C file format.
+ Parameters
+ ----------
+ mat : scipy.sparse.coo_matrix
+ Hi-C contact map.
+ out_prefix : str
+ Output path without extension (the extension is added based on hic_fmt).
+ frags : pandas.DataFrame or None
+ Table of fragments informations.
+ chroms : pandas.DataFrame or None
+ Table of chromosomes / contigs informations.
+ hic_fmt : str
+ Output format. Can be one of graal for graal-compatible COO format, bg2 for
+ 2D bedgraph format, or cool for cooler compatible format.
+ """
+ if hic_fmt == "graal":
+ save_sparse_matrix(mat, out_prefix + ".mat.tsv")
+ try:
+ frags.to_csv(out_prefix + ".frags.tsv", sep="\t", index=False)
+ except AttributeError:
+ if not quiet:
+ logger.warning(
+ "Could not create fragments_list.txt from input files"
+ )
+ try:
+ chroms.to_csv(out_prefix + ".chr.tsv", sep="\t", index=False)
+ except AttributeError:
+ if not quiet:
+ logger.warning(
+ "Could not create info_contigs.txt from input files"
+ )
+ elif hic_fmt == "cool":
+ frag_sizes = frags.end_pos - frags.start_pos
+ size_mad = np.median(frag_sizes - np.median(frag_sizes))
+ bin_type = "variable" if size_mad else "fixed"
+ try:
+ save_cool(
+ out_prefix + ".cool",
+ mat,
+ frags,
+ metadata={"hicstuff": __version__, "bin-type": bin_type},
+ )
+ except NameError:
+ NameError("frags is required to save a cool file")
+ elif hic_fmt == "bg2":
+ try:
+ save_bedgraph2d(mat, frags, out_prefix + ".bg2")
+ except NameError:
+ NameError("frags is required to save a bg2 file")
+ else:
+ raise ValueError("Unknown output format: {0}".format(hic_fmt))
+
+
+def save_bedgraph2d(mat, frags, out_path):
+ """
+ Given a sparse matrix and a corresponding list of fragments, save a file
+ in 2D bedgraph format.
+ Parameters
+ ----------
+ mat : scipy.sparse.coo_matrix
+ The sparse contact map.
+ frags : pandas.DataFrame
+ A structure containing the annotations for each matrix bin. Should
+ correspond to the content of the fragments_list.txt file.
+ """
+
+ mat_df = pd.DataFrame(
+ {"row": mat.row, "col": mat.col, "data": mat.data.astype(int)}
+ )
+ # Merge fragments with matrix based on row indices to annotate rows
+ merge_mat = mat_df.merge(
+ frags, left_on="row", right_index=True, how="left", suffixes=("", "")
+ )
+ # Rename annotations to assign to frag1
+ merge_mat.rename(
+ columns={"chrom": "chr1", "start_pos": "start1", "end_pos": "end1"},
+ inplace=True,
+ )
+ # Do the same operation for cols (frag2)
+ merge_mat = merge_mat.merge(
+ frags,
+ left_on="col",
+ right_index=True,
+ how="left",
+ suffixes=("_0", "_2"),
+ )
+ merge_mat.rename(
+ columns={"chrom": "chr2", "start_pos": "start2", "end_pos": "end2"},
+ inplace=True,
+ )
+ # Select only relevant columns in correct order
+ bg2 = merge_mat.loc[
+ :, ["chr1", "start1", "end1", "chr2", "start2", "end2", "data"]
+ ]
+ bg2.to_csv(out_path, header=None, index=False, sep="\t")
+
+
+def get_pos_cols(df):
+ """Get column names representing chromosome, start and end column
+ from a dataframe. Allows flexible names.
+ Parameters
+ ----------
+ df : pd.DataFrame
+ Returns
+ -------
+ tuple of str:
+ Tuple containing the names of the chromosome, start and end
+ columns in the input dataframe.
+ Examples
+ --------
+ >>> import pandas as pd
+ >>> d = [1, 2, 3]
+ >>> df = pd.DataFrame(
+ ... {'Chromosome': d, 'Start': d, 'End': d, 'species': d}
+ ... )
+ >>> get_pos_cols(df)
+ ('Chromosome', 'Start', 'End')
+ >>> df = pd.DataFrame(
+ ... {'id': d, 'chr': d, 'start_bp': d, 'end_bp': d}
+ ... )
+ >>> get_pos_cols(df)
+ ('chr', 'start_bp', 'end_bp')
+ """
+
+ # Get case insensitive column names
+ cnames = df.columns
+ inames = cnames.str.lower()
+
+ def _col_getter(cols, pat):
+ """Helper to get column index from the start of its name"""
+ mask = cols.str.startswith(pat)
+ idx = np.flatnonzero(mask)[0]
+ return idx
+
+ chrom_col = cnames[_col_getter(inames, "chr")]
+ start_col = cnames[_col_getter(inames, "start")]
+ end_col = cnames[_col_getter(inames, "end")]
+ return chrom_col, start_col, end_col
+
+
+def gc_bins(genome_path, frags):
+ """Generate GC content annotation for bins using input genome.
+ Parameters
+ ----------
+ genome_path : str
+ Path the the genome file in FASTA format.
+ frags : pandas.DataFrame
+ Table containing bin segmentation of the genome.
+ Required columns: chrom, start, end.
+ Returns
+ -------
+ numpy.ndarray of floats:
+ GC content per bin, in the range [0.0, 1.0].
+ """
+ # Grab columns by name (catch start, Start, star_pos, etc)
+ chrom_col, start_col, end_col = get_pos_cols(frags)
+ # Fill the gc array by chromosome
+ gc_bins = np.zeros(frags.shape[0], dtype=float)
+ for rec in SeqIO.parse(genome_path, "fasta"):
+ mask = frags[chrom_col] == rec.id
+ # Define coordinates of each bin
+ starts = frags.loc[mask, start_col]
+ ends = frags.loc[mask, end_col]
+ # Slice chromosome sequence based on bins
+ seqs = [str(rec.seq)[s:e] for s, e in zip(starts, ends)]
+ # Fill GC values for bins in the chromosome
+ idx = np.flatnonzero(mask)
+ gc_bins[idx] = np.array(list(map(SeqUtils.GC, seqs))) / 100.0
+
+ return gc_bins
+
+
+def sort_pairs(in_file, out_file, keys, tmp_dir=None, threads=1, buffer="2G"):
+ """
+ Sort a pairs file in batches using UNIX sort.
+ Parameters
+ ----------
+ in_file : str
+ Path to the unsorted input file
+ out_file : str
+ Path to the sorted output file.
+ keys : list of str
+ list of columns to use as sort keys. Each column can be one of readID,
+ chr1, pos1, chr2, pos2, frag1, frag2. Key priorities are according to
+ the order in the list.
+ tmp_dir : str
+ Path to the directory where temporary files will be created. Defaults
+ to current directory.
+ threads : int
+ Number of parallel sorting threads.
+ buffer : str
+ Buffer size used for sorting. Consists of a number and a unit.
+ """
+ # TODO: Write a pure python implementation to drop GNU coreutils depencency,
+ # could be inspired from: https://stackoverflow.com/q/14465154/8440675
+
+ # Check if UNIX sort version supports parallelism
+ parallel_ok = True
+ sort_ver = sp.Popen(["sort", "--version"], stdout=sp.PIPE)
+ sort_ver = (
+ sort_ver.communicate()[0]
+ .decode()
+ .split("\n")[0]
+ .split(" ")[-1]
+ .split(".")
+ )
+ # If so, specify threads, otherwise don't mention it in the command line
+ try:
+ sort_ver = list(map(int, sort_ver))
+ if sort_ver[0] < 8 or (sort_ver[0] == 8 and sort_ver[1] < 23):
+ logger.warning(
+ "GNU sort version is {0} but >8.23 is required for parallel "
+ "sort. Sorting on a single thread.".format(
+ ".".join(map(str, sort_ver))
+ )
+ )
+ parallel_ok = False
+ # BSD sort has a different format and will throw error upon parsing. It does
+ # not support parallel processes anyway.
+ except ValueError:
+ logger.warning(
+ "Using BSD sort instead of GNU sort, sorting on a single thread."
+ )
+ parallel_ok = False
+
+ key_map = {
+ "readID": "-k1,1d",
+ "chr1": "-k2,2V",
+ "pos1": "-k3,3n",
+ "chr2": "-k4,4V",
+ "pos2": "-k5,5n",
+ "strand1": "-k6,6d",
+ "strand2": "-k7,7d",
+ "frag1": "-k8,8n",
+ "frag2": "-k9,9n",
+ }
+
+ # transform column names to corresponding sort keys
+ try:
+ sort_keys = map(lambda k: key_map[k], keys)
+ except KeyError:
+ print("Unkown column name.")
+ raise
+ # Rewrite header with new sorting order
+ header = get_pairs_header(in_file)
+ with open(out_file, "w") as output:
+ for line in header:
+ if line.startswith("#sorted"):
+ output.write("#sorted: {0}\n".format("-".join(keys)))
+ else:
+ output.write(line + "\n")
+
+ # Sort pairs and append to file.
+ with open(out_file, "a") as output:
+ grep_proc = sp.Popen(["grep", "-v", "^#", in_file], stdout=sp.PIPE)
+ sort_cmd = ["sort", "-S %s" % buffer] + list(sort_keys)
+ if tmp_dir is not None:
+ sort_cmd.append("--temporary-directory={0}".format(tmp_dir))
+ if parallel_ok:
+ sort_cmd.append("--parallel={0}".format(threads))
+ sort_proc = sp.Popen(sort_cmd, stdin=grep_proc.stdout, stdout=output)
+ sort_proc.communicate()
+
+
+def get_pairs_header(pairs):
+ r"""Retrieves the header of a .pairs file and stores lines into a list.
+ Parameters
+ ----------
+ pairs : str or file object
+ Path to the pairs file.
+ Returns
+ -------
+ header : list of str
+ A list of header lines found, in the same order they appear in pairs.
+ Examples
+ --------
+ >>> import os
+ >>> from tempfile import NamedTemporaryFile
+ >>> p = NamedTemporaryFile('w', delete=False)
+ >>> p.writelines(["## pairs format v1.0\n", "#sorted: chr1-chr2\n", "abcd\n"])
+ >>> p.close()
+ >>> h = get_pairs_header(p.name)
+ >>> for line in h:
+ ... print([line])
+ ['## pairs format v1.0']
+ ['#sorted: chr1-chr2']
+ >>> os.unlink(p.name)
+ """
+ # Open file if needed
+ with open(pairs, "r") as pairs:
+ # Store header lines into a list
+ header = []
+ line = pairs.readline()
+ while line.startswith("#"):
+ header.append(line.rstrip())
+ line = pairs.readline()
+
+ return header
+
+
+def reorder_fasta(genome, output=None, threshold=100000):
+ """Reorder and trim a fasta file
+ Sort a fasta file by record lengths, optionally trimming the smallest ones.
+ Parameters
+ ----------
+ genome : str, file or pathlib.Path
+ The genome scaffold file (or file handle)
+ output : str, file or pathlib.Path
+ The output file to write to
+ threshold : int, optional
+ The size below which scaffolds are discarded, by default 100000
+ """
+
+ if output is None:
+ output = "{}_renamed.fa".format(genome.split(".")[0])
+
+ handle = SeqIO.parse(genome, "fasta")
+ handle_to_write = sorted(
+ (len(u) for u in handle if len(u) > threshold), reverse=True
+ )
+ SeqIO.write(handle_to_write, output, "fasta")
+
+
+def rename_genome(genome, output=None, ambiguous=True):
+ """Rename genome and slugify headers
+ Rename genomes according to a simple naming scheme; this
+ is mainly done to avoid special character weirdness.
+ Parameters
+ ----------
+ genome : file, str or pathlib.Path
+ The input genome to be renamed and slugify.
+ output : file, str or pathlib.Path
+ The output genome to be written into. Default is <base>_renamed.fa,
+ where <base> is genome_in without its extension.
+ ambiguous : bool
+ Whether to retain ambiguous non-N bases, otherwise rename them to Ns.
+ Default is True.
+ """
+
+ if output is None:
+ output = "{}_renamed.fa".format(genome.split(".")[0])
+
+ with open(output, "w") as output_handle:
+ for record in SeqIO.parse(genome, "fasta"):
+
+ # Replace hyphens, tabs and whitespace with underscores
+ new_record_id = record.id.replace(" ", "_")
+ new_record_id = new_record_id.replace("-", "_")
+ new_record_id = new_record_id.replace("\t", "_")
+
+ # Remove anything that's weird, i.e. not alphanumeric
+ # or an underscore
+ new_record_id = re.sub("[^_A-Za-z0-9]+", "", new_record_id)
+ header = ">{}\n".format(new_record_id)
+
+ new_seq = re.sub("[^ATGCatgcNn]", "N", str(record.seq))
+
+ output_handle.write(header)
+ output_handle.write("{}\n".format(new_seq))
+
+
+def check_fasta_index(ref, mode="bowtie2"):
+ """
+ Checks for the existence of a bowtie2 or bwa index based on the reference
+ file name.
+ Parameters
+ ----------
+ ref : str
+ Path to the reference genome.
+ mode : str
+ The alignment software used to build the index. bowtie2 or bwa. If any
+ other value is given, the function returns the reference path.
+ Returns
+ -------
+ index : str
+ The bowtie2 or bwa index basename. None if no index was found
+ """
+ ref = pathlib.Path(ref)
+ if mode == "bowtie2":
+ # Bowtie2 should have 6 index files
+ bt2_idx_files = list(ref.parent.glob("{}*bt2*".format(ref.name)))
+ index = None if len(bt2_idx_files) < 6 else bt2_idx_files
+ elif mode == "bwa":
+ refdir = str(ref.parent)
+ refdir_files = os.listdir(refdir)
+ bwa_idx_files = [
+ join(refdir, f)
+ for f in refdir_files
+ if re.search(r".*\.(sa|pac|bwt|ann|amb)$", f)
+ ]
+ index = None if len(bwa_idx_files) < 5 else bwa_idx_files
+ else:
+ index = [ref]
+ if index is not None:
+ # Convert the PosixPath objects to strings and get the longest common prefix to obtain
+ # index basename (without the dot)
+ index = os.path.commonprefix(list(map(str, index))).strip(".")
+ return index
+
+
+def check_is_fasta(in_file):
+ """
+ Checks whether input file is in fasta format.
+
+ Parameters
+ ----------
+ in_file : str
+ Path to the input file.
+ Returns
+ -------
+ bool :
+ True if the input is in fasta format, False otherwise
+ """
+ try:
+ with read_compressed(in_file) as handle:
+ fasta = any(SeqIO.parse(handle, "fasta"))
+ except FileNotFoundError:
+ fasta = False
+
+ return fasta
diff --git a/bin/hicstuff_log.py b/bin/hicstuff_log.py
new file mode 100644
index 0000000..171a566
--- /dev/null
+++ b/bin/hicstuff_log.py
@@ -0,0 +1,89 @@
+#!/usr/bin/env python3
+# coding: utf-8
+
+"""Basic logging
+Basic logging setup with three main handlers (stdout, log file and optionally
+texting with the right API). By default the log file is disabled, but can be
+enabled or changed using set_file_handler.
+"""
+
+
+import logging
+import logging.handlers
+import requests
+import json
+
+TEXT_CREDENTIALS_DEFAULT_PATH = "text_credentials.json"
+
+CURRENT_LOG_LEVEL = logging.INFO
+
+
+logging.captureWarnings(True)
+
+logger = logging.getLogger()
+logger.setLevel(CURRENT_LOG_LEVEL)
+
+logfile_formatter = logging.Formatter(
+ "%(asctime)s :: %(levelname)s :: %(message)s", datefmt="%Y-%m-%d,%H:%M:%S"
+)
+stdout_formatter = logging.Formatter("%(levelname)s :: %(message)s")
+text_formatter = logging.Formatter("%(message)s")
+
+# file_handler = logging.FileHandler("hicstuff.log", "a")
+
+# file_handler.setLevel(logging.INFO)
+# file_handler.setFormatter(logfile_formatter)
+# logger.addHandler(file_handler)
+
+stream_handler = logging.StreamHandler()
+stream_handler.setLevel(logging.DEBUG)
+stream_handler.setFormatter(stdout_formatter)
+logger.addHandler(stream_handler)
+
+
+def set_file_handler(log_path, formatter=logfile_formatter):
+ """Change the file handler for custom log file location"""
+
+ filehandler = logging.FileHandler(log_path, "a")
+ filehandler.setLevel(logging.INFO)
+ filehandler.setFormatter(formatter)
+ for hdlr in logger.handlers[:]: # remove the existing file handlers
+ if isinstance(hdlr, logging.FileHandler):
+ logger.removeHandler(hdlr)
+ logger.addHandler(filehandler) # set the new handler
+
+
+def setup_text_logging(credentials=TEXT_CREDENTIALS_DEFAULT_PATH):
+ """Setup text logging
+ Setup text notifications on errors with a basic API. In order for it to
+ work, a file named 'text_credentials.json' must be in the directory where
+ a command is run. The logger performs a GET request to a text notification
+ API.
+ Parameters
+ ----------
+ credentials : str or pathlib.Path
+ A JSON file with necessary credentials for the GET request
+ """
+
+ with open(credentials) as cred_handle:
+ cred_dict = json.load(cred_handle)
+
+ api_service = cred_dict.pop("api_service")
+
+ class TextHandler(logging.Handler):
+ def emit(self, record):
+ log_entry = self.format(record)
+ cred_dict["msg"] = log_entry
+ return requests.get(api_service, cred_dict)
+
+ sms_handler = TextHandler()
+ sms_handler.setLevel(logging.ERROR)
+ sms_handler.setFormatter(text_formatter)
+
+ logger.addHandler(sms_handler)
+
+
+try:
+ setup_text_logging(TEXT_CREDENTIALS_DEFAULT_PATH)
+except FileNotFoundError:
+ pass
diff --git a/conf/hicstuff.config b/conf/hicstuff.config
index 6e20d06..f26ae40 100644
--- a/conf/hicstuff.config
+++ b/conf/hicstuff.config
@@ -9,6 +9,37 @@ process {
withName: 'BOWTIE2_ALIGNMENT' {
ext.prefix = { "${meta.id}_${meta.chunk}_${meta.mates}" }
}
+
+ withName: 'FRAGMENT_ENZYME' {
+ publishDir = [
+ path: { "${params.outdir}/hicstuff/fragment_enzyme" },
+ mode: 'copy'
+ ]
+ }
+
+ //**********************************************
+ // PREPARE_GENOME
+ withName: 'BOWTIE2_BUILD' {
+ publishDir = [
+ path: { "${params.outdir}/genome/bowtie2" },
+ mode: 'copy'
+ ]
+ }
+
+ withName: 'CUSTOM_GETCHROMSIZES' {
+ publishDir = [
+ path: { "${params.outdir}/genome" },
+ mode: 'copy'
+ ]
+ }
+
+ withName: 'GET_RESTRICTION_FRAGMENTS' {
+ publishDir = [
+ path: { "${params.outdir}/genome" },
+ mode: 'copy'
+ ]
+ }
+
}
params {
diff --git a/modules/local/hicstuff/fragment_enzyme.nf b/modules/local/hicstuff/fragment_enzyme.nf
new file mode 100644
index 0000000..dafa3cf
--- /dev/null
+++ b/modules/local/hicstuff/fragment_enzyme.nf
@@ -0,0 +1,21 @@
+process FRAGMENT_ENZYME {
+ tag "$digestion"
+ label "process_single"
+
+ conda "conda-forge::python=3.9 conda-forge::biopython=1.80 conda-forge::numpy=1.22.3 conda-forge::matplotlib=3.6.3 conda-forge::pandas=1.5.3"
+ container = "lbmc/hicstuff:3.1.3"
+
+ input:
+ val(digestion)
+ tuple val(meta), path(fasta)
+
+ output:
+ tuple val(meta), path("info_contigs.txt") , emit: info_contigs
+ tuple val(meta), path("fragments_list.txt"), emit: fragments_list
+
+ script:
+ """
+ hicstuff_fragments.py -e ${digestion} -i ${fasta}
+ """
+
+}
diff --git a/subworkflows/local/hicstuff_sub.nf b/subworkflows/local/hicstuff_sub.nf
index dfb160b..99516b3 100644
--- a/subworkflows/local/hicstuff_sub.nf
+++ b/subworkflows/local/hicstuff_sub.nf
@@ -1,5 +1,6 @@
include { BOWTIE2_ALIGNMENT } from '../../modules/local/hicstuff/align_bowtie2'
+include { FRAGMENT_ENZYME } from '../../modules/local/hicstuff/fragment_enzyme'
// Paired-end to Single-end
def pairToSingle(row, mates) {
@@ -27,6 +28,8 @@ workflow HICSTUFF_SUB {
reads
index
ligation_site
+ digestion
+ fasta
main:
@@ -39,4 +42,13 @@ workflow HICSTUFF_SUB {
ch_reads,
index.collect()
)
+
+ FRAGMENT_ENZYME(
+ digestion,
+ fasta
+ )
+
+ emit:
+ info_contigs = FRAGMENT_ENZYME.out.info_contigs
+ fragments_list = FRAGMENT_ENZYME.out.fragments_list
}
diff --git a/workflows/hicstuff.nf b/workflows/hicstuff.nf
index 0a760cf..4a69b68 100644
--- a/workflows/hicstuff.nf
+++ b/workflows/hicstuff.nf
@@ -6,9 +6,9 @@ if (params.input) { ch_input = file(params.input) } else { exit 1, 'Input sample
// Digestion parameters
if (params.digestion){
restriction_site = params.digestion ? params.digest[ params.digestion ].restriction_site ?: false : false
- ch_restriction_site = Channel.value(restriction_site)
+ ch_restriction_site = Channel.value(restriction_site) //str seq avec ^ pour cut
ligation_site = params.digestion ? params.digest[ params.digestion ].ligation_site ?: false : false
- ch_ligation_site = Channel.value(ligation_site)
+ ch_ligation_site = Channel.value(ligation_site) //str seq
}else if (params.dnase){
ch_restriction_site = Channel.empty()
ch_ligation_site = Channel.empty()
@@ -37,6 +37,8 @@ workflow HICSTUFF {
HICSTUFF_SUB(
INPUT_CHECK.out.reads,
PREPARE_GENOME.out.index,
- ch_ligation_site
+ ch_ligation_site,
+ params.digestion, //str enzyme
+ ch_fasta
)
}
--
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