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# -*- coding: UTF-8 -*-
#
# This file is part of khmer, https://github.com/dib-lab/khmer/, and is
# Copyright (C) 2010-2015, Michigan State University.
# Copyright (C) 2015-2016, The Regents of the University of California.
#
# Redistribution and use in source and binary forms, with or without
# modification, are permitted provided that the following conditions are
# met:
#
# * Redistributions of source code must retain the above copyright
# notice, this list of conditions and the following disclaimer.
#
# * Redistributions in binary form must reproduce the above
# copyright notice, this list of conditions and the following
# disclaimer in the documentation and/or other materials provided
# with the distribution.
#
# * Neither the name of the Michigan State University nor the names
# of its contributors may be used to endorse or promote products
# derived from this software without specific prior written
# permission.
#
# THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS
# "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT
# LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR
# A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT
# HOLDER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL,
# SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT
# LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE,
# DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY
# THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT
# (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE
# OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.
#
# Contact: khmer-project@idyll.org
# pylint: disable=missing-docstring,protected-access,no-member,invalid-name
from __future__ import print_function
from __future__ import absolute_import
import itertools
import random
import khmer
from khmer.khmer_args import estimate_optimal_with_K_and_f as optimal_fp
from khmer import reverse_complement as revcomp
from . import khmer_tst_utils as utils
import pytest
import screed
# We just define this globally rather than in a module-level fixture,
# as we need it during parameterization and whatnot.
K = 21
class Kmer(str):
def __init__(self, value, pos=0):
self.pos = pos
def __new__(cls, value, pos=0):
if not len(value) == K:
raise ValueError('bad k-mer length')
return str.__new__(cls, value)
def mutate_base(base):
if base in 'AT':
return random.choice('GC')
elif base in 'GC':
return random.choice('AT')
else:
assert False, 'bad base'
def mutate_sequence(sequence, N=1):
sequence = list(sequence)
positions = random.sample(range(len(sequence)), N)
for i in positions:
sequence[i] = mutate_base(sequence[i])
return ''.join(sequence)
def mutate_position(sequence, pos):
sequence = list(sequence)
sequence[pos] = mutate_base(sequence[pos])
return ''.join(sequence)
def get_random_sequence(length, exclude=None):
'''Generate a random (non-looping) nucleotide sequence.
To be non-overlapping, the sequence should not include any repeated
length K-1 k-mers.
Args:
exclude (str): If not None, add the k-mers from this sequence to the
seen set.
Returns:
str: A random non-looping sequence.
'''
seen = set()
def add_seen(kmer):
seen.add(kmer)
seen.add(revcomp(kmer))
if exclude is not None:
for pos in range(0, len(exclude) - K):
add_seen(exclude[pos:pos + K - 1])
seq = [random.choice('ACGT') for _ in range(K - 1)] # do first K-1 bases
add_seen(''.join(seq))
while(len(seq) < length):
next_base = random.choice('ACGT')
next_kmer = ''.join(seq[-K + 2:] + [next_base])
assert len(next_kmer) == K - 1
if (next_kmer) not in seen:
seq.append(next_base)
add_seen(next_kmer)
else:
continue
return ''.join(seq)
def reads(sequence, L=100, N=100, dbg_cover=False):
positions = list(range(len(sequence) - L))
if dbg_cover is True:
for start in range(0, len(sequence), K):
read = sequence[start:start + L]
if len(read) < K:
read = sequence[-L:]
yield read
N -= 1
if N < 0:
return
for i in range(N):
start = random.choice(positions)
yield sequence[start:start + L]
def kmers(sequence):
for i in range(len(sequence) - K + 1):
yield sequence[i:i + K]
def test_mutate_sequence():
for _ in range(100):
assert 'A' not in mutate_sequence('A' * 10, 10)
assert 'T' not in mutate_sequence('T' * 10, 10)
assert 'C' not in mutate_sequence('C' * 10, 10)
assert 'G' not in mutate_sequence('G' * 10, 10)
def test_mutate_position():
assert mutate_position('AAAA', 2) in ['AACA', 'AAGA']
assert mutate_position('TTTT', 2) in ['TTCT', 'TTGT']
assert mutate_position('CCCC', 2) in ['CCAC', 'CCTC']
assert mutate_position('GGGG', 2) in ['GGAG', 'GGTG']
def test_reads():
contigfile = utils.get_test_data('simple-genome.fa')
contig = list(screed.open(contigfile))[0].sequence
for read in reads(contig):
assert read in contig
for read in reads(contig):
assert mutate_sequence(read) not in contig
'''
# GRAPH STRUCTURE FIXTURES
These fixtures emit various graph structures with their corresponding
sequences and important nodes. They take a random sequence fixture and
a graph fixture, then consume sequence and generate k-mers accordingly.
We're using a bespoke but simple language to describe graph structures in the
docstrings of these tests. It is as follows:
o: Node
[x:y]: Node at position in sequence
[x:y]+S: Node at position in sequence with extra base (where S in ACGT)
(Name), ([x:y] Name): Named node, named node at position
→ : Edge
~~: Tandem →o→ repeats
'''
@pytest.fixture(params=['simple-genome.fa'])
def known_sequence(request):
fn = utils.get_test_data(request.param)
return list(screed.open(fn))[0].sequence
@pytest.fixture(params=list(range(500, 1600, 500)),
ids=lambda val: '(L={0})'.format(val))
def random_sequence(request):
def get(exclude=None):
return get_random_sequence(request.param, exclude=exclude)
return get
@pytest.fixture(params=[khmer.Nodegraph, khmer.Countgraph],
ids=['(Type=Nodegraph)', '(Type=Countgraph)'])
def graph(request):
num_kmers = 50000
des_fp = 0.00001
args = optimal_fp(num_kmers, des_fp)
print('Graph Params:', args)
return request.param(K, args.htable_size, args.num_htables)
def hdn_counts(sequence, graph):
'''Get the degree distribution of nodes with degree more than 2.
'''
hdns = {}
for kmer in kmers(sequence):
d = graph.kmer_degree(kmer)
if d > 2:
hdns[d] = hdns.get(d, 0) + 1
return hdns
@pytest.fixture
def linear_structure(request, graph, random_sequence):
'''Sets up a simple linear path graph structure.
sequence
[0]→o→o~~o→o→[-1]
'''
sequence = random_sequence()
graph.consume(sequence)
# Check for false positive neighbors in our graph
# Mark as an expected failure if any are found
if hdn_counts(sequence, graph):
request.applymarker(pytest.mark.xfail)
return graph, sequence
@pytest.fixture(params=[K * 2, -K * 2],
ids=['(Where={0})'.format(i) for i in ['Start', 'End']])
def right_tip_structure(request, graph, random_sequence):
'''
Sets up a graph structure like so:
([S+1:S+K]+B tip)
sequence ↗
[0]→o→o~~o→(L)→([S:S+K] HDN)→(R)→o→o→o~~o→[-1]
Where S is the start position of the high degreen node (HDN).
That is, it has a single branch at the Sth K-mer.
'''
sequence = random_sequence()
S = request.param
if S < 0:
S = len(sequence) + S
# the HDN
HDN = Kmer(sequence[S:S + K], pos=S)
# left of the HDN
L = Kmer(sequence[S - 1:S - 1 + K], pos=S - 1)
# right of the HDN
R = Kmer(sequence[S + 1:S + 1 + K], pos=S + 1)
# the branch kmer
tip = Kmer(mutate_position(R, -1),
pos=R.pos)
graph.consume(sequence)
graph.count(tip)
# Check for false positive neighbors and mark as expected failure if found
if hdn_counts(sequence, graph) != {3: 1}:
request.applymarker(pytest.mark.xfail)
return graph, sequence, L, HDN, R, tip
@pytest.fixture(params=[K * 2, -K * 2],
ids=['(Where={0})'.format(i) for i in ['Start', 'End']])
def right_double_fork_structure(request, linear_structure, random_sequence):
'''
Sets up a graph structure like so:
branch
([S+1:S+K]+B)→o~~o→o
core_sequence ↗
[0]→o→o~~o→(L)→([S:S+K] HDN)→(R)→o→o→o~~o→[-1]
Where S is the start position of the high degreen node (HDN)
and B is the mutated base starting the branch.
'''
graph, core_sequence = linear_structure
print('\nCore Len:', len(core_sequence))
branch_sequence = random_sequence(exclude=core_sequence)
print('Branch len:', len(branch_sequence))
# start position of the HDN
S = request.param
if S < 0:
S = len(core_sequence) + S
# the HDN
HDN = Kmer(core_sequence[S:S + K], pos=S)
# left of the HDN
L = Kmer(core_sequence[S - 1:S - 1 + K], pos=S - 1)
# right of the HDN
R = Kmer(core_sequence[S + 1:S + 1 + K], pos=S + 1)
# the branch sequence, mutated at position S+1
branch_start = core_sequence[:R.pos] + mutate_position(R, -1)
branch_sequence = branch_start + branch_sequence
graph.consume(core_sequence)
graph.consume(branch_sequence)
# Check for false positive neighbors and mark as expected failure if found
core_hdns = hdn_counts(core_sequence, graph)
branch_hdns = hdn_counts(branch_sequence, graph)
# the core and branch sequences should each have exactly
# ONE node of degree 3 (HDN)
if core_hdns != {3: 1} or branch_hdns != {3: 1}:
print(core_hdns, branch_hdns)
request.applymarker(pytest.mark.xfail)
return graph, core_sequence, L, HDN, R, branch_sequence
@pytest.fixture
def right_triple_fork_structure(request, right_double_fork_structure,
random_sequence):
'''
Sets up a graph structure like so:
top_branch
([:S+1]+B)→o~~o→o
core_sequence ↗
[0]→o→o~~o→(L)→([S:S+K] HDN)→(R)→o→o→o~~o→[-1]
↘
([:S+1]+B)→o~~o→o
bottom_branch
Where S is the start position of the high degreen node (HDN).
'''
graph, core_sequence, L, HDN, R, top_sequence = right_double_fork_structure
bottom_branch = random_sequence(exclude=core_sequence + top_sequence)
print(len(core_sequence), len(top_sequence), len(bottom_branch))
# the branch sequence, mutated at position S+1
# choose a base not already represented at that position
bases = {'A', 'C', 'G', 'T'}
mutated = random.choice(list(bases - {R[-1], top_sequence[R.pos + K - 1]}))
bottom_sequence = core_sequence[:HDN.pos + K] + mutated + bottom_branch
graph.consume(bottom_sequence)
# Check for false positive neighbors and mark as expected failure if found
core_hdns = hdn_counts(core_sequence, graph)
top_hdns = hdn_counts(top_sequence, graph)
bottom_hdns = hdn_counts(bottom_sequence, graph)
# the core, top, and bottom sequences should each have exactly
# ONE node of degree 4 (HDN)
if not (core_hdns == top_hdns == bottom_hdns == {4: 1}):
print(core_hdns, top_hdns, bottom_hdns)
request.applymarker(pytest.mark.xfail)
return graph, core_sequence, L, HDN, R, top_sequence, bottom_sequence
@pytest.fixture(params=[K * 2, -K * 2],
ids=['(Where={0})'.format(i) for i in ['Start', 'End']])
def left_tip_structure(request, graph, random_sequence):
'''
Sets up a graph structure like so:
branch
(B+[S:S+K-1] tip)
↘ sequence
[0]→o~~o→(L)→([S:S+K] HDN)→(R)→o→o~~o→[-1]
Where S is the start position of the HDN.
'''
sequence = random_sequence()
S = request.param
if S < 0:
S = len(sequence) + S
tip = Kmer(mutate_position(sequence[S - 1:S - 1 + K], 0),
pos=S - 1 + K)
HDN = Kmer(sequence[S:S + K], pos=S)
L = Kmer(sequence[S - 1:S - 1 + K], pos=S - 1)
R = Kmer(sequence[S + 1:S + 1 + K], pos=S + 1)
graph.consume(sequence)
graph.count(tip)
# Check for false positive neighbors and mark as expected failure if found
if hdn_counts(sequence, graph) != {3: 1}:
request.applymarker(pytest.mark.xfail)
return graph, sequence, L, HDN, R, tip
@pytest.fixture(params=[K * 2, -K * 2],
ids=['(Where={0})'.format(i) for i in ['Start', 'End']])
def left_double_fork_structure(request, linear_structure, random_sequence):
'''
Sets up a graph structure like so:
o→o~~o→(B+[S:S+K-1])
↘ core_sequence
[0]→o→o~~o→(L)→([S:S+K] HDN)→(R)→o→o→o~~o→[-1]
Where S is the start position of the high degreen node (HDN).
'''
graph, core_sequence = linear_structure
branch_sequence = random_sequence(exclude=core_sequence)
# start position of the HDN
S = request.param
if S < 0:
S = len(core_sequence) + S
# the HDN
HDN = Kmer(core_sequence[S:S + K], pos=S)
# left of the HDN
L = Kmer(core_sequence[S - 1:S - 1 + K], pos=S - 1)
# right of the HDN
R = Kmer(core_sequence[S + 1:S + 1 + K], pos=S + 1)
# the branch sequence, mutated at position 0 in L,
# whih is equivalent to the K-1 prefix of HDN prepended with a new base
branch_start = mutate_position(L, 0)
branch_sequence = branch_sequence + \
branch_start + core_sequence[L.pos + K:]
graph.consume(core_sequence)
graph.consume(branch_sequence)
# Check for false positive neighbors and mark as expected failure if found
core_hdns = hdn_counts(core_sequence, graph)
branch_hdns = hdn_counts(branch_sequence, graph)
# the core and branch sequences should each have exactly
# ONE node of degree 3 (HDN)
if not (core_hdns == branch_hdns == {3: 1}):
request.applymarker(pytest.mark.xfail)
return graph, core_sequence, L, HDN, R, branch_sequence
@pytest.fixture(params=[K * 2, (-K * 2) - 2],
ids=['(Where={0})'.format(i) for i in ['Start', 'End']])
def snp_bubble_structure(request, linear_structure):
'''
Sets up a graph structure resulting from a SNP (Single Nucleotide
Polymorphism).
(HDN_L[1:]+SNP)→o~~o→(SNP+)
↗ ↘
o~~([S:S+K] HDN_L) ([S+K+1:S+2K+1] HDN_R)~~o
↘ ↗
(HDN_L[1:]+W)→o~~o~~o→(W+)
Where S is the start position of HDN directly left of the SNP (HDN_L),
SNP is the mutated base, and W is the wildtype (original) base.
Of course, W and SNP could be interchanged here, we don't actually
know which is which ;)
Note our parameterization: we need a bit more room from the ends,
so we bring the rightmost SNP a tad left.
'''
graph, wildtype_sequence = linear_structure
S = request.param
if S < 0:
S = len(wildtype_sequence) + S
snp_sequence = mutate_position(wildtype_sequence, S + K)
HDN_L = Kmer(wildtype_sequence[S:S + K], pos=S)
HDN_R = Kmer(wildtype_sequence[S + K + 1:S + 2 * K + 1], pos=S + K + 1)
graph.consume(wildtype_sequence)
graph.consume(snp_sequence)
# Check for false positive neighbors and mark as expected failure if found
w_hdns = hdn_counts(wildtype_sequence, graph)
snp_hdns = hdn_counts(snp_sequence, graph)
if not (w_hdns == snp_hdns == {3: 2}):
print(w_hdns, snp_hdns)
print(HDN_L, HDN_R)
print(wildtype_sequence[HDN_L.pos + K + 1])
print(snp_sequence[HDN_L.pos + K + 1])
request.applymarker(pytest.mark.xfail)
return graph, wildtype_sequence, snp_sequence, HDN_L, HDN_R
@pytest.fixture(params=[2, 3, 4, 5, 6, 7, 8])
def tandem_repeat_structure(request, linear_structure):
graph, sequence = linear_structure
tandem_repeats = sequence * request.param
graph.consume(tandem_repeats)
if hdn_counts(tandem_repeats, graph):
request.applymarker(pytest.mark.xfail)
return graph, sequence, tandem_repeats
@pytest.fixture
def circular_linear_structure(request, linear_structure):
graph, sequence = linear_structure
sequence += sequence
if hdn_counts(sequence, graph):
request.applymarker(pytest.mark.xfail)
return graph, sequence
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