菜单
Batch effect correction for CRISPR screens. Covers normalization across batches, technical replicate handling, and batch-aware analysis. Use when combining screens from multiple batches or correcting systematic technical variation.
| name | bio-crispr-screens-batch-correction |
| description | Batch effect correction for CRISPR screens. Covers normalization across batches, technical replicate handling, and batch-aware analysis. Use when combining screens from multiple batches or correcting systematic technical variation. |
| tool_type | python |
| primary_tool | scipy |
Reference examples tested with: DESeq2 1.42+, MAGeCK 0.5+, matplotlib 3.8+, numpy 1.26+, pandas 2.2+, scikit-learn 1.4+, scipy 1.12+
Before using code patterns, verify installed versions match. If versions differ:
pip show <package> then help(module.function) to check signaturesIf code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
"Correct batch effects in my CRISPR screens" → Normalize and harmonize sgRNA count data across screen batches to remove systematic technical variation while preserving biological signal.
scipy/sklearn for median normalization and batch correctionmageck test with batch-aware designGoal: Remove systematic library-size differences between batches.
Approach: Scale each sample within a batch so that sample medians match a global median, correcting for sequencing depth variation.
import numpy as np
import pandas as pd
from scipy import stats
def median_normalize(counts_df, batch_column='batch'):
'''Normalize counts to median within each batch.'''
normalized = counts_df.copy()
guide_columns = [c for c in counts_df.columns if c not in [batch_column, 'gene', 'guide']]
for batch in counts_df[batch_column].unique():
batch_mask = counts_df[batch_column] == batch
batch_data = counts_df.loc[batch_mask, guide_columns]
sample_medians = batch_data.median(axis=0)
global_median = sample_medians.median()
scale_factors = global_median / sample_medians
normalized.loc[batch_mask, guide_columns] = batch_data * scale_factors
return normalized
counts_df = pd.read_csv('screen_counts.csv')
normalized = median_normalize(counts_df, 'batch')
def size_factor_normalize(counts_df, reference='geometric_mean'):
'''DESeq2-style size factor normalization.'''
guide_cols = [c for c in counts_df.columns if c.startswith('sample_')]
counts = counts_df[guide_cols].values
counts_nonzero = np.where(counts == 0, np.nan, counts)
if reference == 'geometric_mean':
log_counts = np.log(counts_nonzero)
geometric_mean = np.exp(np.nanmean(log_counts, axis=1))
else:
geometric_mean = counts_nonzero.mean(axis=1)
ratios = counts_nonzero / geometric_mean[:, np.newaxis]
size_factors = np.nanmedian(ratios, axis=0)
normalized_counts = counts / size_factors
normalized_df = counts_df.copy()
normalized_df[guide_cols] = normalized_counts
return normalized_df, size_factors
normalized, size_factors = size_factor_normalize(counts_df)
print('Size factors:', size_factors)
def quantile_normalize(counts_df, guide_cols=None):
'''Quantile normalization across samples.'''
if guide_cols is None:
guide_cols = [c for c in counts_df.columns if c.startswith('sample_')]
data = counts_df[guide_cols].values.copy()
sorted_data = np.sort(data, axis=0)
mean_values = sorted_data.mean(axis=1)
ranks = np.argsort(np.argsort(data, axis=0), axis=0)
normalized = mean_values[ranks]
result = counts_df.copy()
result[guide_cols] = normalized
return result
qn_counts = quantile_normalize(counts_df)
def normalize_to_controls(counts_df, control_genes, method='median'):
'''Normalize using non-targeting or negative control guides.'''
guide_cols = [c for c in counts_df.columns if c.startswith('sample_')]
is_control = counts_df['gene'].isin(control_genes)
control_data = counts_df.loc[is_control, guide_cols]
if method == 'median':
control_values = control_data.median(axis=0)
elif method == 'mean':
control_values = control_data.mean(axis=0)
elif method == 'sum':
control_values = control_data.sum(axis=0)
reference = control_values.median()
scale_factors = reference / control_values
normalized = counts_df.copy()
normalized[guide_cols] = counts_df[guide_cols] * scale_factors
return normalized, scale_factors
nontargeting = counts_df[counts_df['gene'].str.startswith('NonTargeting')]['gene'].unique()
normalized, factors = normalize_to_controls(counts_df, nontargeting)
Goal: Remove batch effects using empirical Bayes adjustment while preserving biological signal.
Approach: Log-transform counts, apply pyCombat with a batch vector, and back-transform to count space.
def combat_correction(counts_df, batch_vector, guide_cols=None):
'''ComBat batch correction for count data.'''
from combat.pycombat import pycombat
if guide_cols is None:
guide_cols = [c for c in counts_df.columns if c.startswith('sample_')]
data = counts_df[guide_cols].values.T
log_data = np.log2(data + 1)
corrected = pycombat(log_data, batch_vector)
corrected_counts = np.power(2, corrected) - 1
corrected_counts = np.maximum(corrected_counts, 0)
result = counts_df.copy()
result[guide_cols] = corrected_counts.T
return result
batches = [1, 1, 1, 2, 2, 2]
corrected = combat_correction(counts_df, batches)
def batch_aware_lfc(counts_df, treatment_cols, control_cols, batch_vector):
'''Calculate LFC accounting for batch structure.'''
batches = np.unique(batch_vector)
lfc_by_batch = []
for batch in batches:
batch_treat = [c for c, b in zip(treatment_cols, batch_vector) if b == batch and c in treatment_cols]
batch_ctrl = [c for c, b in zip(control_cols, batch_vector) if b == batch and c in control_cols]
if len(batch_treat) == 0 or len(batch_ctrl) == 0:
continue
treat_mean = counts_df[batch_treat].mean(axis=1)
ctrl_mean = counts_df[batch_ctrl].mean(axis=1)
batch_lfc = np.log2((treat_mean + 1) / (ctrl_mean + 1))
lfc_by_batch.append(batch_lfc)
combined_lfc = pd.concat(lfc_by_batch, axis=1).mean(axis=1)
lfc_var = pd.concat(lfc_by_batch, axis=1).var(axis=1)
return combined_lfc, lfc_var
def check_replicate_correlation(counts_df, sample_cols, replicate_groups):
'''Check correlation between replicates.'''
correlations = []
for group, replicates in replicate_groups.items():
if len(replicates) < 2:
continue
for i in range(len(replicates)):
for j in range(i+1, len(replicates)):
r1, r2 = replicates[i], replicates[j]
if r1 in sample_cols and r2 in sample_cols:
log_r1 = np.log2(counts_df[r1] + 1)
log_r2 = np.log2(counts_df[r2] + 1)
corr, pval = stats.pearsonr(log_r1, log_r2)
correlations.append({
'group': group,
'rep1': r1,
'rep2': r2,
'pearson_r': corr,
'pvalue': pval
})
return pd.DataFrame(correlations)
replicate_groups = {
'treatment_batch1': ['sample_1', 'sample_2'],
'treatment_batch2': ['sample_4', 'sample_5'],
'control_batch1': ['sample_3'],
'control_batch2': ['sample_6']
}
corr_df = check_replicate_correlation(counts_df, counts_df.columns[3:], replicate_groups)
print(corr_df)
Goal: Quantify batch effect magnitude to determine whether correction is needed.
Approach: Run PCA on log-transformed counts, compute between-batch vs within-batch variance ratio, and assess whether batch structure dominates the first principal components.
def batch_qc_metrics(counts_df, batch_vector, sample_cols):
'''Calculate batch-related QC metrics.'''
from sklearn.decomposition import PCA
from scipy.spatial.distance import pdist
log_counts = np.log2(counts_df[sample_cols].values.T + 1)
pca = PCA(n_components=min(5, len(sample_cols)))
pcs = pca.fit_transform(log_counts)
batch_labels = np.array(batch_vector)
unique_batches = np.unique(batch_labels)
if len(unique_batches) > 1:
batch_means = [pcs[batch_labels == b].mean(axis=0) for b in unique_batches]
batch_separation = np.mean(pdist(batch_means))
within_batch_var = np.mean([pcs[batch_labels == b].var() for b in unique_batches])
between_batch_var = np.var(batch_means, axis=0).sum()
batch_effect_ratio = between_batch_var / (within_batch_var + 1e-10)
else:
batch_separation = 0
batch_effect_ratio = 0
return {
'batch_separation': batch_separation,
'batch_effect_ratio': batch_effect_ratio,
'pca_variance_explained': pca.explained_variance_ratio_,
'n_batches': len(unique_batches)
}
qc = batch_qc_metrics(counts_df, [1,1,1,2,2,2], sample_cols)
print(f"Batch effect ratio: {qc['batch_effect_ratio']:.2f}")
import matplotlib.pyplot as plt
def plot_batch_effect(counts_df, batch_vector, sample_cols, output_file):
'''Visualize batch effects with PCA.'''
from sklearn.decomposition import PCA
log_counts = np.log2(counts_df[sample_cols].values.T + 1)
pca = PCA(n_components=2)
pcs = pca.fit_transform(log_counts)
fig, ax = plt.subplots(figsize=(8, 6))
for batch in np.unique(batch_vector):
mask = np.array(batch_vector) == batch
ax.scatter(pcs[mask, 0], pcs[mask, 1], label=f'Batch {batch}', s=100)
ax.set_xlabel(f'PC1 ({pca.explained_variance_ratio_[0]:.1%})')
ax.set_ylabel(f'PC2 ({pca.explained_variance_ratio_[1]:.1%})')
ax.legend()
ax.set_title('PCA - Batch Effects')
plt.tight_layout()
plt.savefig(output_file, dpi=150)
plt.close()
plot_batch_effect(counts_df, [1,1,1,2,2,2], sample_cols, 'batch_pca.png')
Treats a ctDNA assay as a molecule-counting experiment at the Poisson edge and builds its analytical-validation case the measurement-science way. Covers the genome-equivalent currency (~330 haploid copies/ng), the lambda = input_GE x VAF sampling ceiling (lambda>=3 for ~95% detection), the error-suppression ladder (raw NGS ~1e-3 -> single-strand UMI ~1e-4/1e-5 -> duplex <1e-7), the CLSI EP17 LoB/LoD/LoD95/LoQ framework, the per-locus-vs-panel-integrated LoD distinction that lets bespoke MRD reach ppm, contrived/SEQC2 reference standards, and honest LoD reporting conditioned on input mass + consensus depth + replicate detection rate. Use when stating or trusting a sensitivity claim, designing a dilution-series validation, deciding how many genome equivalents are needed at a target VAF, choosing a single-locus vs panel-integrated LoD, or auditing a "detects 0.1% VAF" claim.
Decides how to preprocess plasma cfDNA sequencing data so the recoverable signal survives - library-prep-aware fragment expectations (dsDNA vs ssDNA/adaptase prep), UMI/duplex consensus with fgbio (ExtractUmisFromBam, GroupReadsByUmi --strategy paired for duplex, CallMolecularConsensusReads vs CallDuplexConsensusReads, FilterConsensusReads min-reads "total s1 s2"), the align->group->consensus->RE-align ordering, and the cfDNA dedup trap where naive coordinate dedup collapses nucleosome-coincident independent molecules. Covers when single-strand consensus suffices vs when duplex is mandatory, the singleton/sensitivity tax at low input, and reading the insert-size histogram as a pre-analytical QC instrument. Use when processing plasma cfDNA reads before fragmentomics, ctDNA mutation calling, or tumor-fraction estimation.
Infer orthologous genes and gene families across species using OrthoFinder3 (HOG-based phylogenetic orthology), SonicParanoid2, Broccoli, ProteinOrtho, OMA / FastOMA hierarchical orthologous groups, eggNOG-mapper, JustOrthologs, and TOGA whole-genome-alignment orthology. Use when building single-copy ortholog sets for phylogenomics, classifying co-orthologs and in/out-paralogs after gene duplication, propagating functional annotation via orthology with awareness of the ortholog conjecture, distinguishing speciation from duplication via gene-tree species-tree reconciliation, computing Quest-for-Orthologs benchmark performance, or running synteny-aware ortholog detection in WGD-affected lineages.
Batch effect correction for CRISPR screens covering ComBat empirical-Bayes, RUV, SVA, control-sgRNA normalization, and the model-based alternative of including batch as a covariate in MAGeCK MLE or Chronos. Covers screen-specific batch sources (passage cohort, library lot, infection day, sequencing run, Cas9 lot, FBS lot), PCA + variance-decomposition diagnostic to decide if correction is needed, when correction harms biology by over-correcting condition into batch, limma removeBatchEffect for visualization-only correction, and relationship to multi-condition design matrices. Use when combining screens for joint analysis, when passage cohort confounds biology, when DepMap-style panels need Chronos with batch covariates, when picking ComBat vs RUV, or when correction harms biology and should be replaced with explicit covariate modeling.
Detects somatic mutations in circulating tumor DNA, treating low-VAF detection as a signal-versus-noise problem set by error suppression and molecules sampled, not by the choice of caller. Distinguishes de novo CALLING (scanning a panel for unknown variants, bounded by per-locus error and multiple testing) from tumor-informed DETECTION (tracking a pre-specified variant set, where panel integration reaches single-ppm). Covers VarDict and Mutect2 for de novo calling, UMI-aware callers, and a pysam-based known-variant VAF tracker, with matched-WBC subtraction as the mandatory defense against clonal hematopoiesis (the dominant false positive). Use when calling or tracking tumor mutations from plasma cfDNA, setting a VAF threshold, or deciding whether a low-VAF call is tumor versus CHIP.
Detects A/B chromatin compartments from balanced Hi-C contact matrices via eigenvector decomposition of the distance-normalized, Pearson-correlated cis matrix with cooltools (eigs_cis), then orients (phases) the compartment eigenvector against a GC or gene-density track so the active (A) sign is not arbitrary. Covers the eigenvector-is-a-choice problem (per-arm view_df to remove the centromere gradient; picking the eigenvector by max correlation with activity, not by eigenvalue), GC phasing with bioframe.frac_gc, resolution choice (100kb-1Mb), saddle plots and saddle_strength for compartmentalization strength, the cohesin-loss-strengthens-compartments result, subcompartments (SNIPER/Calder/dcHiC), and cross-condition compartment switching. Use when calling A/B compartments, computing E1/eigenvectors, phasing the eigenvector, building saddle plots, choosing a compartment resolution, quantifying compartment strength, or comparing compartmentalization across conditions.