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tools.py
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import numpy as np
import seaborn as sns
from datetime import datetime
import pickle
from scipy import sparse
from sklearn.decomposition import PCA
from scipy.special import xlogy
import sys
sys.path.append('/tmp/FIt-SNE-master/') ### ADAPT PATH TO FIt-SNE REPO AS NEEDED
from fast_tsne import fast_tsne
sys.path.append('../../libs/glmpca-py/') ### ADAPT PATH TO glmpca-py REPO AS NEEDED
from glmpca import glmpca
sys.path.append('../../libs/rna-seq-tsne/') ### ADAPT PATH TO rna-seq-tsne REPO AS NEEDED
import rnaseqTools
def add_labels(dataset, xdata,ydata,example_genes,textoffsets,lines,ax):
for example_gene,textoffset,line in zip(example_genes,textoffsets,lines):
gene_idx = dataset['genes'] == example_gene
gene_position = np.array([xdata[gene_idx],ydata[gene_idx]]).flatten()
text_position = np.array([gene_position[0]*10**textoffset[0],gene_position[1]+textoffset[1]]).flatten()
ax.text(*text_position,example_gene.capitalize(),horizontalalignment='center')
if line:
line_start = line[0]
line_end = line[1]
line_x = [text_position[0]*10**line_start[0],gene_position[0]*10**line_end[0]]
line_y = [text_position[1]+line_start[1],gene_position[1]+line_end[1]]
ax.plot(line_x,line_y,'k',linewidth=2)
def add_largedot_legend(ax,loc,kwargs={}):
lgnd = ax.legend(loc=loc,frameon=True,**kwargs)
for l in lgnd.legendHandles:
l._sizes = [30]
def compute_marginals(counts):
'''compute depths per cell (ns) and relative expression fractions per gene (ps)'''
ns = np.sum(counts,axis=1)
ps = np.sum(counts,axis=0)
ps = ps / np.sum(ps)
return np.squeeze(np.array(ns)), np.squeeze(np.array(ps))
def compute_means(counts):
'''compute gene means and their min, max and 50 logspaced values covering this range'''
means = np.squeeze(np.array(np.mean(counts,axis=0)))
mean_min = min(means)
mean_max = max(means)
mean_range = np.logspace(np.log10(mean_min),np.log10(mean_max))
return means, mean_min, mean_max, mean_range
def get_glmpca_timestamps(date_string):
date_object = datetime.strptime(date_string, "%Y-%m-%d %H:%M:%S.%f")
return date_object
def kobak_tsne(data,name='',n_PCs=50,init=None,do_pca=True,seed=42,perplexities=None):
if do_pca:
X,PCAinit = PCA_sklearn(data,n_PCs=n_PCs,seed=seed)
print('pca is done')
else:
X=data
if init is None and do_pca:
init = PCAinit
n = X.shape[0]
if perplexities is None:
perplexities = [30, int(n/100)]
print(perplexities)
learning_rate= n/12
tsne = dict(perplexity_list=perplexities, initialization=init, learning_rate=learning_rate, seed=seed)
print('tSNE input shape:',X.shape)
tsne['coords'] = fast_tsne(X, perplexity_list=perplexities, initialization=init, learning_rate=learning_rate,seed=seed)
filename = 'tsne/tsne_%s.pickle' % (name)
with open(filename, "wb") as file:
pickle.dump(tsne, file, protocol=4)
print('tSNE output shape:',tsne['coords'].shape)
return tsne
def kobak_tsne_w_exag(data,name='',n_PCs=50,init=None,do_pca=True,exag=4,start_late_exag_iter=250,seed=42,print_info=False):
pass
if do_pca:
X,PCAinit = PCA_sklearn(data,n_PCs=n_PCs,seed=seed)
print('pca is done')
else:
X=data
if init is None:
init = PCAinit
perplexities = [30]
learning_rate=X.shape[0]/12
tsne = dict(perplexity_list=perplexities, initialization=init, learning_rate=learning_rate, seed=seed, exag=exag,start_late_exag_iter=start_late_exag_iter)
if print_info:
print('tSNE of X with',X.shape)
print('init with ',init.shape)
print('perplexity_list',perplexities)
print('late_exag_coeff',exag)
print('start_late_exag_iter',start_late_exag_iter)
print('learning_rate',learning_rate)
print('seed',seed)
tsne['coords'] = fast_tsne(X, perplexity_list=perplexities, late_exag_coeff=exag, start_late_exag_iter=start_late_exag_iter,initialization=init, learning_rate=learning_rate,seed=seed)
filename = 'tsne/tsne_%s.pickle' % (name)
with open(filename, "wb") as file:
pickle.dump(tsne, file, protocol=4)
return tsne
def monitor_progress(gene_id,n_genes,print_time_every=1000,print_dot_every=25):
if np.mod(gene_id,print_time_every)==0:
print('')
print('##',datetime.now().time(),'##',gene_id,'of',n_genes,'genes fit',end='')
if np.mod(gene_id,print_dot_every)==0:
print('.',end='')
def normalize_and_scale(counts,scale_mode='user_provided_scale',scale=1):
'''Depth-normalizes and scales count matrix'''
depths = np.squeeze(np.array(np.sum(counts,axis=1)))
if scale_mode=='median':
scale = np.median(depths)
elif scale_mode=='user_provided_scale':
#using provided scale value
pass
return (counts.T/depths).T * scale
def sqrt_lazy(counts):
'''Depth-normalizes, scales by median depth and then takes the square root.'''
normalized = normalize_and_scale(counts,scale_mode='median')
return np.sqrt(normalized)
def log_transform(counts,scale_mode,scale=1):
'''Depth-normalizes, scales by median me'''
normalized = normalize_and_scale(counts,scale_mode=scale_mode,scale=scale)
return np.log1p(normalized)
def PCA_sklearn(data,n_PCs,seed):
X = np.array(data)
pca = PCA(n_components=n_PCs,random_state=seed)
X = pca.fit_transform(X)
print('variance explained by top%u PCs: %u %%' % (n_PCs, sum(pca.explained_variance_ratio_)*100))
#scale values relative to absolute max value
X = X / np.max(np.abs(X))
#use top2 PCs for intialisation
PCAinit = X[:,:2] / np.std(X[:,0]) * .0001
return X, PCAinit
def PCA_sklearn_timing(data,n_PCs,seed):
X = np.array(data)
pca = PCA(n_components=n_PCs,random_state=seed)
X = pca.fit_transform(X)
def pearson_residuals(counts, theta, clipping=True):
'''Computes analytical residuals for NB model with a fixed theta, clipping outlier residuals to sqrt(N)'''
counts_sum0 = np.sum(counts, axis=0, keepdims=True)
counts_sum1 = np.sum(counts, axis=1, keepdims=True)
counts_sum = np.sum(counts)
#get residuals
mu = counts_sum1 @ counts_sum0 / counts_sum
z = (counts - mu) / np.sqrt(mu + mu**2/theta)
#clip to sqrt(n)
if clipping:
n = counts.shape[0]
z[z > np.sqrt(n)] = np.sqrt(n)
z[z < -np.sqrt(n)] = -np.sqrt(n)
return z
def deviance_residuals(x, theta,mu=None):
'''Computes deviance residuals for NB model with a fixed theta'''
if mu is None:
counts_sum0 = np.sum(x, axis=0, keepdims=True)
counts_sum1 = np.sum(x, axis=1, keepdims=True)
counts_sum = np.sum(x)
#get residuals
mu = counts_sum1 @ counts_sum0 / counts_sum
def remove_negatives(sqrt_term):
negatives_idx = sqrt_term < 0
if np.any(negatives_idx):
n_negatives = np.sum(negatives_idx)
print('Setting %u negative sqrt term values to 0 (%f%%)' % (n_negatives,n_negatives/np.product(sqrt_term.shape)))
sqrt_term[negatives_idx] = 0
if np.isinf(theta): ### POISSON
x_minus_mu = x-mu
sqrt_term = 2 * (xlogy(x,x/mu) - x_minus_mu ) #xlogy(x,x/mu) computes xlog(x/mu) and returns 0 if x=0
remove_negatives(sqrt_term)
dev = np.sign(x_minus_mu) * np.sqrt(sqrt_term)
else: ### NEG BIN
x_plus_theta = x+theta
sqrt_term = 2 * ( xlogy(x,x/mu) - (x_plus_theta) * np.log(x_plus_theta/(mu+theta)) ) #xlogy(x,x/mu) computes xlog(x/mu) and returns 0 if x=0
remove_negatives(sqrt_term)
dev = np.sign(x-mu) * np.sqrt(sqrt_term)
return dev
def prepare_largest_batch(dataset,extra_keys=[]):
###add counts and clusters for largest batch only to the dataset
#find largest batch
batch_ids, batch_counts = np.unique(dataset['batches'],return_counts=True)
largest_batch_id_idx = np.argmax(batch_counts)
largest_batch_id = batch_ids[largest_batch_id_idx]
largest_batch_idx = dataset['batches'] == largest_batch_id
#slice accordingly and clean out rare genes
dataset['counts'],dataset['genes'] = remove_rare_genes(dataset['counts'][largest_batch_idx,:],dataset['genes'],5)
dataset['clusters'] = dataset['clusters'][largest_batch_idx]
dataset['labelshort'] = dataset['labelshort'] + '_largestBatch'
dataset['batches'] = dataset['batches'][largest_batch_idx]
for k in extra_keys:
dataset[k] = dataset[k][largest_batch_idx]
def remove_rare_genes(counts,genes,minimum_detected_cells_per_gene):
if type(counts) in [sparse.csr.csr_matrix, sparse.csc.csc_matrix]:
#remove zero genes
nonzero_genes_idx = np.array(counts.sum(axis=0)).flatten() > 0
counts = counts[:,nonzero_genes_idx]
genes = genes[nonzero_genes_idx]
#count nonzero entries per gene
nonzero_coords = counts.nonzero()
n_nonzero = counts.count_nonzero()
is_nonzero = sparse.csc_matrix((np.ones(n_nonzero),nonzero_coords))
detected_cells_per_gene = np.array(is_nonzero.sum(axis=0)).flatten()
keep_genes = detected_cells_per_gene >= minimum_detected_cells_per_gene
counts_kept = counts[:,keep_genes]
genes_kept = genes[keep_genes]
print('Of',len(detected_cells_per_gene),'total genes, returning',sum(keep_genes),'genes that are detected in %u or more cells.' % (minimum_detected_cells_per_gene))
print('Output shape:', counts_kept.shape)
return counts_kept,np.array(genes_kept)
else:
#remove zero genes
nonzero_genes_idx = np.sum(counts,axis=0) > 0
counts = counts[:,nonzero_genes_idx]
genes = genes[nonzero_genes_idx]
#remove genes that are detected in less then n cells
nonzero = counts > 0
cells_per_gene = np.sum(nonzero,axis=0)
include_genes = cells_per_gene >= minimum_detected_cells_per_gene
counts_kept = counts[:,include_genes]
genes_kept = genes[include_genes]
print('Of',len(cells_per_gene),'total genes, returning',sum(include_genes),'genes that are detected in %u or more cells.' % (minimum_detected_cells_per_gene))
print('Output shape:', counts_kept.shape)
return counts_kept,genes_kept
def run_glmpca(counts,fam,theta = 100, penalty = 1, optimize_nb_theta=True, maxIter=1000, eps=0.0001, n_PCs=50, seed=42, dataset_label=''):
'''Wrapper around GLM PCA by Will Townes: applies GLM PCA with given settings and saves results as pickle'''
np.random.seed(seed)
ctl = {"maxIter":maxIter, "eps":eps, "optimizeTheta":optimize_nb_theta}
if maxIter==1000 and eps == 0.0001:
ctl_str=''
else:
ctl_str='_maxIter%u_eps%s' % (maxIter,eps)
starttime = str(datetime.now())
res = glmpca.glmpca(counts.T,n_PCs,fam=fam,nb_theta=theta,verbose=True,penalty=penalty,ctl=ctl)
endtime = str(datetime.now())
res['starttime']=starttime
res['endtime']=endtime
if fam=='nb':
res['nb_theta']=res['glmpca_family'].nb_theta
_ = res.pop('glmpca_family')
if fam=='poi':
path = 'glmpca_results/%sglmpca-py_%s_penalty%u%s.pickle' % (dataset_label,fam,penalty,ctl_str)
elif optimize_nb_theta:
path = 'glmpca_results/%sglmpca-py_%s_penalty%u%s.pickle' % (dataset_label,fam,penalty,ctl_str)
else:
path = 'glmpca_results/%sglmpca-py_%s_fixedTheta%u_penalty%u%s.pickle' % (dataset_label,fam,theta,penalty,ctl_str)
print('Saving at', path)
with open(path,'wb') as f:
pickle.dump(res,f)
def pickle_sanity_results(filename, show_progress=False):
with open(filename, "r") as f:
linedata = []
prefixes = []
if show_progress:
print('reading in genes line by line:')
for i,line in enumerate(f.readlines()):
if show_progress:
print(i,end=' ')
if i==0:
headers = np.array(line.replace('GeneID\t','').replace('\n','').split('\t'))
continue
prefix = 'Gene_%u\t' % (i-1)
assert prefix == line[:len(prefix)]
prefixes.append(line[:len(prefix)].replace('\t',''))
ldat=np.fromstring(line[len(prefix):], sep = "\t")
linedata.append(ldat)
print('\n')
print('stacking lines')
sanity_ltqs=np.vstack(linedata).T
print('saving pickle')
with open(filename+'.pickle','wb') as f:
pickle.dump(sanity_ltqs,f)
### Code extended from https://github.com/theislab/scanpy/pull/1715 for Deviance residuals
## imports for scanpy env
import warnings
from typing import Optional, Union
import numpy as np
import pandas as pd
import scipy.sparse as sp_sparse
from anndata import AnnData
from scanpy import logging as logg
from scanpy._settings import settings, Verbosity
from scanpy._utils import sanitize_anndata, check_nonnegative_integers, view_to_actual
from scanpy.get import _get_obs_rep, _set_obs_rep
from scanpy._compat import Literal
from scanpy.preprocessing._utils import _get_mean_var
from scanpy.preprocessing._distributed import materialize_as_ndarray
from scanpy.preprocessing._simple import filter_genes
def highly_variable_residuals(
adata: AnnData,
layer: Optional[str] = None,
n_top_genes: int = 1000,
batch_key: Optional[str] = None,
theta: float = 100,
clip: Optional[float] = None,
chunksize: int = 100,
check_values: bool = True,
subset: bool = False,
inplace: bool = True,
residual_type: str = 'pearson',
debug=False,
) -> Optional[pd.DataFrame]:
"""\
See `highly_variable_genes`.
Returns
-------
Depending on `inplace` returns calculated metrics (:class:`~pd.DataFrame`)
or updates `.var` with the following fields:
highly_variable
boolean indicator of highly-variable genes.
means
means per gene.
variances
variances per gene.
residual_variances
Pearson residual variance per gene. Averaged in the case of multiple
batches.
highly_variable_rank
Rank of the gene according to residual variance, median rank in the
case of multiple batches.
highly_variable_nbatches : int
If batch_key is given, this denotes in how many batches genes are
detected as HVG.
highly_variable_intersection : bool
If batch_key is given, this denotes the genes that are highly variable
in all batches.
"""
view_to_actual(adata)
X = _get_obs_rep(adata, layer=layer)
computed_on = layer if layer else 'adata.X'
# Check for raw counts
if check_values and (check_nonnegative_integers(X) == False):
warnings.warn(
"`flavor='pearson_residuals'` expects raw count data, but non-integers were found.",
UserWarning,
)
if batch_key is None:
batch_info = np.zeros(adata.shape[0], dtype=int)
else:
batch_info = adata.obs[batch_key].values
n_batches = len(np.unique(batch_info))
# Get pearson residuals for each batch separately
residual_gene_vars = []
for batch in np.unique(batch_info):
adata_subset = adata[batch_info == batch]
# Filter out zero genes
with settings.verbosity.override(Verbosity.error):
nonzero_genes = filter_genes(adata_subset, min_cells=1, inplace=False)[0]
adata_subset = adata_subset[:, nonzero_genes]
if layer is not None:
X_batch = adata_subset.layers[layer]
else:
X_batch = adata_subset.X
# Prepare clipping
if clip is None:
n = X_batch.shape[0]
clip = np.sqrt(n)
if clip < 0:
raise ValueError("Pearson residuals require `clip>=0` or `clip=None`.")
if sp_sparse.issparse(X_batch):
sums_genes = np.sum(X_batch, axis=0)
sums_cells = np.sum(X_batch, axis=1)
sum_total = np.sum(sums_genes).squeeze()
else:
sums_genes = np.sum(X_batch, axis=0, keepdims=True)
sums_cells = np.sum(X_batch, axis=1, keepdims=True)
sum_total = np.sum(sums_genes)
# Compute pearson residuals in chunks
residual_gene_var = np.empty((X_batch.shape[1]))
for start in np.arange(0, X_batch.shape[1], chunksize):
stop = start + chunksize
X_dense = X_batch[:, start:stop].toarray()
mu = np.array(sums_cells @ sums_genes[:, start:stop] / sum_total)
if residual_type == 'pearson':
residuals = (X_dense - mu) / np.sqrt(mu + mu ** 2 / theta)
residuals = np.clip(residuals, a_min=-clip, a_max=clip)
elif residual_type == 'deviance':
residuals = deviance_residuals(X_dense,theta,mu)
residual_gene_var[start:stop] = np.var(residuals, axis=0)
# Add 0 values for genes that were filtered out
zero_gene_var = np.zeros(np.sum(~nonzero_genes))
residual_gene_var = np.concatenate((residual_gene_var, zero_gene_var))
# Order as before filtering
idxs = np.concatenate((np.where(nonzero_genes)[0], np.where(~nonzero_genes)[0]))
residual_gene_var = residual_gene_var[np.argsort(idxs)]
residual_gene_vars.append(residual_gene_var.reshape(1, -1))
residual_gene_vars = np.concatenate(residual_gene_vars, axis=0)
# Get cutoffs and define hvgs per batch
residual_gene_vars_sorted = np.sort(residual_gene_vars, axis=1)
cutoffs_per_batch = residual_gene_vars_sorted[:, -n_top_genes]
highly_variable_per_batch = np.greater_equal(
residual_gene_vars.T, cutoffs_per_batch
).T
# Merge hvgs across batches
highly_variable_nbatches = np.sum(highly_variable_per_batch, axis=0)
highly_variable_intersection = highly_variable_nbatches == n_batches
# Get rank per gene within each batch
# argsort twice gives ranks, small rank means most variable
ranks_residual_var = np.argsort(np.argsort(-residual_gene_vars, axis=1), axis=1)
ranks_residual_var = ranks_residual_var.astype(np.float32)
ranks_residual_var[ranks_residual_var >= n_top_genes] = np.nan
ranks_masked_array = np.ma.masked_invalid(ranks_residual_var)
# Median rank across batches,
# ignoring batches in which gene was not selected
medianrank_residual_var = np.ma.median(ranks_masked_array, axis=0).filled(np.nan)
means, variances = materialize_as_ndarray(_get_mean_var(X))
df = pd.DataFrame.from_dict(
dict(
means=means,
variances=variances,
residual_variances=np.mean(residual_gene_vars, axis=0),
highly_variable_rank=medianrank_residual_var,
highly_variable_nbatches=highly_variable_nbatches,
highly_variable_intersection=highly_variable_intersection,
)
)
df = df.set_index(adata.var_names)
# Sort genes by how often they selected as hvg within each batch and
# break ties with median rank of residual variance across batches
df.sort_values(
['highly_variable_nbatches', 'highly_variable_rank'],
ascending=[False, True],
na_position='last',
inplace=True,
)
df['highly_variable'] = False
df.highly_variable.iloc[:n_top_genes] = True
# TODO: following line raises a pandas warning
# (also for flavor = seurat and cellranger..)
df = df.loc[adata.var_names]
if inplace or subset:
adata.uns['hvg'] = {'flavor': 'pearson_residuals', 'computed_on': computed_on}
logg.hint(
'added\n'
' \'highly_variable\', boolean vector (adata.var)\n'
' \'highly_variable_rank\', float vector (adata.var)\n'
' \'highly_variable_nbatches\', int vector (adata.var)\n'
' \'highly_variable_intersection\', boolean vector (adata.var)\n'
' \'means\', float vector (adata.var)\n'
' \'variances\', float vector (adata.var)\n'
' \'residual_variances\', float vector (adata.var)'
)
adata.var['highly_variable'] = df['highly_variable'].values
adata.var['highly_variable_rank'] = df['highly_variable_rank'].values
adata.var['means'] = df['means'].values
adata.var['variances'] = df['variances'].values
adata.var['residual_variances'] = df['residual_variances'].values.astype(
'float64', copy=False
)
if batch_key is not None:
adata.var['highly_variable_nbatches'] = df[
'highly_variable_nbatches'
].values
adata.var['highly_variable_intersection'] = df[
'highly_variable_intersection'
].values
if subset:
adata._inplace_subset_var(df['highly_variable'].values)
else:
if batch_key is None:
df = df.drop(
['highly_variable_nbatches', 'highly_variable_intersection'], axis=1
)
return df