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__version__ = '1.4.0'
import argparse
from concurrent.futures import ThreadPoolExecutor, ProcessPoolExecutor
import itertools
import functools
import h5py
import numpy as np
import os
from libscrappy import ffi, lib
ftype = np.float32
size_ftype = np.dtype(ftype).itemsize
vsize = 4 # SSE vector length
def _none_if_null(p):
# convert cffi NULL to None
if p == ffi.NULL:
p = None
return p
def _gsp():
# allowing up to 9mer, we can have up to base7 and be unique
alpha_len = range(4,8)
kmer_len = range(1,10)
pairs = [(a, k) for a, k in itertools.product(alpha_len, kmer_len)]
state_lookup = {a**k: (a, k) for a, k in pairs}
assert len(state_lookup) == len(pairs) # check all are unique
def guess_state_properties(nstate):
"""Find likely kmer length and alphabet size from transducer state-space size.
:param nstate: number of states in transducer.
:returns: alphabet size, kmer length.
"""
nkmers = nstate - 1 # for stay
return state_lookup[nkmers]
return guess_state_properties
guess_state_properties = _gsp()
class RawTable(object):
def __init__(self, data, start=0, end=None):
"""Representation of a scrappie `raw_table`.
:param data: `nd.array` containing raw data.
..note:: The class stores a reference to a contiguous numpy array of
the correct type to be passed to the extension library. The class
provides safety against the original data being garbage collected.
To obtain an up-to-date (possibly trimmed and scaled) copy of the
data use `raw_table.data(as_numpy=True)`.
"""
if end is None:
end = len(data)
self._data = np.ascontiguousarray(data.astype(ftype, order='C', copy=True))
rt = ffi.new('raw_table *')
rt.uuid = ffi.NULL
rt.n = len(self._data)
rt.start = start
rt.end = end
rt.raw = ffi.cast("float *", ffi.from_buffer(self._data))
self._rt = rt[0]
def data(self, as_numpy=False):
"""Current data as either C object or realised numpy copy.
:param as_numpy: If True, return a numpy copy of the current data. If
currently selected range empty, a (0,) shape array is returned.
"""
if as_numpy:
return np.copy(self._data[self.start:self.end])
else:
return self._rt
@property
def start(self):
"""Currently set start sample."""
return self._rt.start
@property
def end(self):
"""Currently set end sample."""
return self._rt.end
def trim(self, start=200, end=10, varseg_chunk=100, varseg_thresh=0.0):
"""Trim data.
:param start: lower bound on number of samples to trim from start
:param end: lower bound on number of samples to trim from end
:param varseg_chunk: chunk size for analysing variance of signal
:param varseq_thresh: quantile to be calculated to use for thresholding
"""
self._rt = _trim_raw(
self._rt, start=start, end=end,
varseg_chunk=varseg_chunk, varseg_thresh=varseg_thresh
)
return self
def scale(self):
"""Normalize data using med/mad scaling."""
self._rt = _scale_raw(self._rt)
return self
def _trim_raw(rt, start=200, end=10, varseg_chunk=100, varseg_thresh=0.0):
"""Trim a `raw_table`.
:param rt: a `raw_table`.
:param start: lower bound on number of samples to trim from start
:param end: lower bound on number of samples to trim from end
:param varseg_chunk: chunk size for analysing variance of signal
:param varseg_thresh: quantile to be calculated to use for thresholding
:returns: new scrappie raw data structure.
"""
rt = _none_if_null(lib.trim_raw_by_mad(rt, varseg_chunk, varseg_thresh))
if rt is not None:
rt.start = rt.start + start if (rt.n - rt.start) > start else rt.n
rt.end = rt.end - end if (rt.end > end) else 0
if rt.start >= rt.end:
rt.start, rt.end = 0, 0
# n.b. at this point the equivalent trim_and_segment_raw() from
# scrappie_common.c frees the data array.
return rt
def _scale_raw(rt):
"""Scale a `raw_table` in place.
:param rt: `raw_table` to scale`.
:returns: input (this is solely to be explicit, `rt` is modified in place).
"""
lib.medmad_normalise_array(rt.raw + rt.start, rt.end - rt.start)
return rt
class ScrappyMatrix(object):
def __init__(self, scrappy_matrix):
"""Container to manage lifetime of a bare scrappie_matrix.
Can be initialised by a pointer to a `scrappy_matrix` or a numpy `ndarray`.
"""
if isinstance(scrappy_matrix, np.ndarray):
self._data = _numpy_to_scrappy_matrix(scrappy_matrix)
elif isinstance(scrappy_matrix, ffi.CData):
self._data = scrappy_matrix
else:
raise TypeError('ScrappyMatrix can only be contructed from a '
'`scrappy_matrix pointer or `ndarray`.')
def __del__(self):
_free_matrix(self._data)
@property
def shape(self):
"""Tuple (columns, rows)
.. note:: The rows value is of the realised array. The actual data is
stored padded for ease of use with SSE vectors.
"""
return self._data.nc, self._data.nr
def data(self, as_numpy=False, sloika=True):
"""Current data as either C object or realised numpy copy. In the
latter case, padding due to SSE vector use is removed.
:param as_numpy: If True, return a numpy copy of the current data.
:param sloika: return sloika compatible posterior matrix (stay is
first state). Only valid when `as_numpy` is `True`.
:returns: a contiguous `np.ndarray` of shape (blocks, states).
"""
if as_numpy:
return _scrappie_to_numpy(self._data, sloika=sloika)
else:
return self._data
def __getitem__(self, slice):
return ScrappyMatrixView(self, slice)
def __len__(self):
return self.shape[0]
class ScrappyMatrixView(ScrappyMatrix):
def __init__(self, scrappy_matrix_obj, slice):
"""Container to provide view of `ScrappyMatrix`.
Won't free underlying data upon garbage collection.
:param scrappy_matrix_obj: `ScrappyMatrix` instance.
:param slice: slice
"""
n_elems = scrappy_matrix_obj.shape[0]
start = slice.start if slice.start is not None else 0
stop = slice.stop if slice.stop is not None else n_elems
if slice.step is not None and slice.step != 1:
raise ValueError('stride must be 1 for a view')
if start > stop:
raise IndexError('start should be smaller than stop')
for i in start, stop:
if i > n_elems:
raise IndexError('index {} is out of bounds for axis 0 with size {}'.format(i, n_elems))
attrs = ['nr', 'nrq', 'nc', 'stride', 'data']
init_data = [getattr(scrappy_matrix_obj._data, a) for a in attrs]
self._data = ffi.new("scrappie_matrix", init=init_data)
self._data.data.f += start * self._data.stride
self._data.nc = stop - start
def __del__(self):
pass # This is a view, we don't want underlying data garbage collected
def _numpy_to_scrappy_matrix(numpy_array):
"""Convert a `ndarray` to a bare `scrappie_matrix`"""
nc = numpy_array.shape[0]
nr = numpy_array.shape[1]
data = np.ascontiguousarray(numpy_array.astype(ftype, order='C', copy=False))
buf = ffi.cast("float *", data.ctypes.data)
return lib.mat_from_array(buf, nr, nc)
def _free_matrix(matrix):
"""Free a `scrappie_matrix`.
:param matrix: a scrappie matrix
:returns: `None`
"""
lib.free_scrappie_matrix(matrix)
def _scrappie_to_numpy(matrix, sloika=True):
"""Convert a `scrappie_matrix` to a numpy array. Removes padding due to
SSE vectors and optionally reorders states.
:param matrix: a `scrappie_matrix` to use as source data.
:param sloika: return sloika compatible matrix (stay is first state).
:returns: a contiguous `np.ndarray` of shape (blocks, states).
..note:: a copy of the data is made, so the input matrix should be freed
at the earliest convenience with `_free_matrix`.
"""
np_matrix = np.frombuffer(ffi.buffer(
matrix.data.f, size_ftype * vsize * matrix.nrq * matrix.nc),
dtype=ftype
).reshape(matrix.nc, vsize * matrix.nrq)
nblock, nstate = matrix.nc, matrix.nr
np_matrix = np_matrix[:, :nstate]
# sloika requires stay state first
if sloika:
p1 = np_matrix[:, nstate - 1:nstate]
p2 = np_matrix[:, 0:nstate - 1]
np_matrix = np.hstack((p1, p2))
np_matrix = np.ascontiguousarray(np_matrix)
return np_matrix
def calc_post(rt, model='rgrgr_r94', min_prob=1e-6, log=True, tempW=1.0, tempb=1.0):
"""Run a network network to obtain class probabilities.
:param rt: a `RawTable`.
:param min_prob: minimum bound of probabilites.
:param log: return log-probabilities.
:returns: a `ScrappyMatrix`
"""
if not log and model == 'rnnrf_r94':
raise ValueError("Returning non-log transformed matrix not supported for model type 'rnnrf_r94'.")
if not isinstance(rt, RawTable):
raise TypeError('`rt` should be a RawTable.')
try:
network = _models_[model]
except KeyError:
raise KeyError("Model type '{}' not recognised.".format(model))
else:
matrix = _none_if_null(network(rt.data(), min_prob, tempW, tempb, log))
if matrix is None:
raise RuntimeError('An unknown error occurred during posterior calculation.')
else:
return ScrappyMatrix(matrix)
def decode_post(post, model='rgrgr_r94', **kwargs):
"""Decode a posterior to retrieve basecall, score, and states. This
function merely dispatches to a relevant function governed by the model.
:param post: a `ScrappyMatrix` containing matrix to be decoded.
:param model: model type.
:param **kwargs: See the functions `_decode_...` for kwargs
relevant to each model.
:returns: tuple containing (call, score, call positions per raw block).
"""
if not isinstance(post, ScrappyMatrix):
raise TypeError('`post` should be a ScrappyMatrix.')
try:
decoder = _decoders_[model]
except KeyError:
raise KeyError("Model type '{}' not recognised.".format(model))
else:
return decoder(post, **kwargs)
def _decode_post(post, stay_pen=0.0, skip_pen=0.0, local_pen=2.0, use_slip=False):
"""Decode a posterior using Viterbi algorithm for transducer.
:param post: a `ScrappyMatrix` containing transducer posteriors.
:param stay_pen: penalty for staying.
:param skip_pen: penalty for skipping a base.
:param local_pen: penalty for local basecalling.
:param use_slip: allow slipping (movement more than 2 bases).
:returns: tuple containing (call, score, call positions per raw block).
"""
nblock, nstate = post.shape
path = ffi.new("int[{}]".format(nblock + 1))
score = lib.decode_transducer(
post.data(), stay_pen, skip_pen, local_pen,
path, use_slip
)
pos = np.zeros(nblock + 1, dtype=np.int32)
p_pos = ffi.cast("int *", pos.ctypes.data)
basecall = lib.overlapper(path, nblock + 1, nstate - 1, p_pos)
return ffi.string(basecall).decode(), score, pos
def _decode_post_crf(post):
"""Decode a posterior using Viterbi algorithm for conditional random field.
:param post: a `ScrappyMatrix` containing CRF transitions.
:returns: tuple containing (basecall, score, call positions per raw data block).
"""
nblock, nstate = post.shape
path = ffi.new("int[{}]".format(nblock + 1))
score = lib.decode_crf(post.data(), path)
pos = np.ascontiguousarray(np.zeros(nblock + 1, dtype=np.int32))
p_pos = ffi.cast("int *", ffi.from_buffer(pos))
basecall = lib.crfpath_to_basecall(path, nblock, p_pos)
return ffi.string(basecall).decode(), score, pos
# Network and decoder functions used above
_models_ = {
'rgrgr_r94': lib.nanonet_rgrgr_r94_posterior,
'rgrgr_r941': lib.nanonet_rgrgr_r941_posterior,
'rgrgr_r10': lib.nanonet_rgrgr_r10_posterior,
'rnnrf_r94': lib.nanonet_rnnrf_r94_transitions,
}
_squiggle_models_ = {
'squiggle_r94': lib.squiggle_r94,
'squiggle_r94_rna': lib.squiggle_r94_rna,
'squiggle_r10': lib.squiggle_r10,
}
_decoders_ = {
'rgrgr_r94': _decode_post,
'rgrgr_r941': _decode_post,
'rgrgr_r10': _decode_post,
'rnnrf_r94': _decode_post_crf,
}
def get_model_stride(model):
"""Obtain the stride length of a model from its name.
:param model: model name:
:returns: the model stride.
"""
stride = lib.get_raw_model_stride_from_string(model.encode())
if stride == -1:
raise ValueError("Invalid scrappie model '{}'.")
return stride
def basecall_raw(data, model='rgrgr_r94', with_base_probs=False, **kwargs):
"""Basecall from raw data in a numpy array to demonstrate API.
:param data: `ndarray` containing raw signal data.
:param model: model to use in calculating basecall.
:param with_base_probs: calculate per-block base (ACGT-) probabilities.
:param kwargs: kwargs passed to `decode_post`.
:returns: tuple containing: (basecall, score, per-block call positions
data start index, data end index, base probs). The last item will
be `None` for `with_base_probs == False`.
"""
if with_base_probs and model != 'rnnrf_r94':
ValueError("Base probabilities can only be returned for model 'rnnrf_r94'.")
raw = RawTable(data)
raw.trim().scale()
post = calc_post(raw, model, log=True)
seq, score, pos = decode_post(post, model, **kwargs)
base_probs = None
if with_base_probs:
base_post = lib.posterior_crf(post.data())
base_probs = _scrappie_to_numpy(base_post, sloika=False)
_free_matrix(base_post)
return seq, score, pos, raw.start, raw.end, base_probs
def sequence_to_squiggle(sequence, model='squiggle_r94', rescale=False):
"""Simulate a squiggle from a base sequence.
:param sequence: base sequence to model.
:param model: model to use in simulating squiggle.
:param rescale:
:param as_numpy: return a numpy array rather than a `scrappie_matrix`.
:returns: a simulated squiggle as either a `ScrappyMatrix` or an ndarray.
"""
seq_len = len(sequence)
seq = _none_if_null(lib.encode_bases_to_integers(sequence.encode(), seq_len, 1))
if seq is None:
raise RuntimeError('An unknown error occurred whilst encoding sequence.')
try:
squiggle_model = _squiggle_models_[model]
except KeyError:
raise KeyError("Squiggle model type '{}' not recognised.".format(model))
else:
squiggle = _none_if_null(squiggle_model(seq, seq_len, rescale))
if squiggle is None:
raise RuntimeError('An unknown error occurred whilst generating squiggle.')
return ScrappyMatrix(squiggle)
def map_signal_to_squiggle(data, sequence, model='squiggle_r94', rate=1.0,
back_prob=0.0, local_pen=2.0, skip_pen=5000.0,
min_score=5.0):
"""Align a squiggle to a sequence using a simulated squiggle.
:param data: `ndarray` containing raw signal data.
:param sequence: base sequence to which to align data.
:param model: model to use in simulating squiggle.
:param rate: rate of translocation relative to squiggle model
:param back_prob: probability of backward movement.
:param local_pen: penalty for local alignment.
:param skip_pen: penalty for skipping position in sequence.
:param min_score: floor on match score.
:returns: tuple containing (alignment score, alignment path)
"""
raw = RawTable(data)
raw.trim().scale()
squiggle = sequence_to_squiggle(sequence, model=model)
path = np.ascontiguousarray(np.zeros(raw._rt.n, dtype=np.int32))
p_path = ffi.cast("int32_t *", ffi.from_buffer(path))
score = lib.squiggle_match_viterbi(raw.data(), rate, squiggle.data(), back_prob,
local_pen, skip_pen, min_score, p_path)
return score, path
def map_post_to_sequence(post, sequence, stay_pen=0, skip_pen=0, local_pen=4.0,
viterbi=False, path=False, bands=None):
"""Block-based local-global alignment of a squiggle to a sequence using
either Forward or Viterbi algorithm. For the latter the Viterbi path can
optionally be calculated.
:param post: a `ScrappyMatrix` containing log-probabilities (as from
`calc_post`).
:param sequence: a base sequence which to map.
:param stay_pen: penalty for zero-state movement from one block to next.
:param skip_pen: penalty for two-state movement from one block to next.
:param local_pen: penalty for local alignment through blocks
:param viterbi: use Viterbi algorithm rather than forward.
:param path: calculate alignment path (only valid for `viterbi==True`
and `bands==None`).
:param bands: two sequences containing lower and upper extremal allowed
positions for each block. Should be length corresponding to number
of blocks of `post`. If a single number is given, a diagonal band with
width 2 * `bands` * #states / #blocks will be used. If `None` is given
banding is not used (a full DP matrix is evaluated).
:returns: (score, path), (or (None, *) in the case of failure).
..note:: if `viterbi`==False or `path`==False, the returned path will
be `None`.
"""
if path and not viterbi:
raise ValueError('Cannot calulate path with `viterbi==False`.')
if not isinstance(post, ScrappyMatrix):
raise TypeError('`post` should be a ScrappyMatrix.')
nblock, nstate = post.shape
alpha_len, kmer_len = guess_state_properties(nstate)
seq_len = len(sequence) - kmer_len + 1
p_seq = _none_if_null(lib.encode_bases_to_integers(sequence.encode(), len(sequence), kmer_len))
if p_seq is None:
raise RuntimeError('An unknown error occurred whilst encoding sequence.')
if viterbi and path:
path_data = np.zeros(nblock, dtype=np.int32)
p_path = ffi.cast("int *", ffi.from_buffer(path_data))
else:
path_data = None
p_path = ffi.NULL
if bands is None:
if viterbi:
score = lib.map_to_sequence_viterbi(
post.data(), stay_pen, skip_pen, local_pen, p_seq, seq_len, p_path)
else:
score = lib.map_to_sequence_forward(
post.data(), stay_pen, skip_pen, local_pen, p_seq, seq_len)
else:
if isinstance(bands, int):
# create a monotonic diagonal band
gradient = seq_len / nblock
bands = 2 * bands * gradient
hband = bands / 2
bands = [np.ascontiguousarray(np.array(x, dtype=np.uintp)) for x in (
[(max(0, x * gradient - hband)) for x in range(nblock)],
[(min(seq_len, x * gradient + hband)) for x in range(nblock)]
)]
elif len(bands) == 2:
bands = [
np.ascontiguousarray(x, dtype=np.uintp) for x in bands]
else:
raise ValueError('`bands` should be `None`, an integer, or length 2.')
p_poslow, p_poshigh = (
ffi.cast("size_t *", ffi.from_buffer(x)) for x in bands)
if not lib.are_bounds_sane(p_poslow, p_poshigh, nblock, seq_len):
raise ValueError('Supplied banding structure is not valid.')
if viterbi:
func = lib.map_to_sequence_viterbi_banded
else:
func = lib.map_to_sequence_forward_banded
score = func(
post.data(), stay_pen, skip_pen, local_pen, p_seq, seq_len, p_poslow, p_poshigh)
score = _none_if_null(score)
if score is None:
raise RuntimeError('An unknown error occurred during alignment.')
return score, path_data
def _raw_gen(filelist):
for fname in filelist:
with h5py.File(fname, 'r') as h:
pass
try:
data = None
with h5py.File(fname, 'r') as h:
base = 'Raw/Reads'
read_name = list(h[base].keys())[0]
data = h['{}/{}/Signal'.format(base, read_name)][()].astype(np.float32)
meta = h['/UniqueGlobalKey/channel_id'].attrs
raw_unit = meta['range'] / meta['digitisation']
data = (data + meta['offset']) * raw_unit
except:
raise RuntimeError('Failed to read signal data from {}.'.format(fname))
else:
yield os.path.basename(fname), data
def _basecall():
# Entry point for testing/demonstration.
parser = argparse.ArgumentParser(
description="Basecall a single .fast5.",
formatter_class=argparse.ArgumentDefaultsHelpFormatter
)
parser.add_argument('fast5', nargs='+',
help='path to .fast5s to basecall.')
parser.add_argument('model', choices=list(_models_.keys()),
help='Choice of model.')
parser.add_argument('--threads', default=None, type=int,
help='Number of threads to use.')
parser.add_argument('--process', action='store_true',
help='Use ProcesPool rather than ThreadPool.')
args = parser.parse_args()
worker = functools.partial(basecall_raw, model=args.model)
if args.threads is None:
for fname, data in _raw_gen(args.fast5):
seq, score, _, start, end, _ = worker(data)
print(">{} {} {}-{}\n{}".format(fname, score, start, end, seq))
else:
iter0, iter1 = itertools.tee(_raw_gen(args.fast5))
Executor = ProcessPoolExecutor if args.process else ThreadPoolExecutor
with Executor(max_workers=args.threads) as executor:
datas = (x[1] for x in iter0)
fnames = (x[0] for x in iter1)
results = executor.map(worker, datas)
for fname, (seq, score, _, start, end, _) in zip(fnames, results):
print(">{} {} {}-{}\n{}".format(fname, score, start, end, seq))
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