bio-crispr-screens-screen-qc

by FreedomIntelligence

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SKILL.md About

---
name: bio-crispr-screens-screen-qc
description: Quality control for pooled CRISPR screens. Covers library representation, read distribution, replicate correlation, and essential gene recovery. Use when assessing screen quality before hit calling or diagnosing poor screen performance.
tool_type: python
primary_tool: pandas
---

## Version Compatibility

Reference examples tested with: MAGeCK 0.5+, matplotlib 3.8+, numpy 1.26+, pandas 2.2+, scikit-learn 1.4+, seaborn 0.13+

Before using code patterns, verify installed versions match. If versions differ:
- Python: `pip show <package>` then `help(module.function)` to check signatures

If code throws ImportError, AttributeError, or TypeError, introspect the installed
package and adapt the example to match the actual API rather than retrying.

# CRISPR Screen Quality Control

**"Check the quality of my CRISPR screen"** → Assess screen quality through library representation, Gini index, replicate correlation, and essential gene recovery metrics before hit calling.
- Python: `pandas` + `matplotlib` for QC metrics and diagnostic plots

## Load Count Data

```python
import pandas as pd
import numpy as np
import matplotlib.pyplot as plt
import seaborn as sns

# Load MAGeCK count output
counts = pd.read_csv('screen.count.txt', sep='\t', index_col=0)
genes = counts['Gene']
count_matrix = counts.drop('Gene', axis=1)

print(f'sgRNAs: {len(count_matrix)}')
print(f'Genes: {genes.nunique()}')
print(f'Samples: {count_matrix.columns.tolist()}')
```

## Library Representation

**Goal:** Assess whether the sgRNA library is adequately represented across all samples.

**Approach:** Count zero-count and low-count sgRNAs per sample, flagging samples where dropout exceeds quality thresholds (>1% zero-count is warning, >5% is failure).

```python
# Zero-count sgRNAs per sample
zero_counts = (count_matrix == 0).sum()
zero_pct = zero_counts / len(count_matrix) * 100

print('Zero-count sgRNAs per sample:')
for sample, pct in zero_pct.items():
    status = 'OK' if pct < 1 else 'WARNING' if pct < 5 else 'FAIL'
    print(f'  {sample}: {pct:.2f}% [{status}]')

# Low-count sgRNAs (<30 reads)
low_counts = (count_matrix < 30).sum()
low_pct = low_counts / len(count_matrix) * 100
print('\nLow-count sgRNAs (<30 reads):')
for sample, pct in low_pct.items():
    print(f'  {sample}: {pct:.2f}%')
```

## Read Distribution (Gini Index)

**Goal:** Quantify how evenly reads are distributed across sgRNAs within each sample.

**Approach:** Calculate the Gini index (0 = perfect equality, 1 = complete inequality) from sorted non-zero counts, where values below 0.2 indicate good uniformity.

```python
def gini_index(x):
    '''Calculate Gini index (0=perfect equality, 1=complete inequality)'''
    x = np.sort(x[x > 0])
    n = len(x)
    cumx = np.cumsum(x)
    return (n + 1 - 2 * np.sum(cumx) / cumx[-1]) / n

gini_values = count_matrix.apply(gini_index)
print('\nGini index per sample (lower is better, <0.2 ideal):')
for sample, gini in gini_values.items():
    status = 'OK' if gini < 0.2 else 'WARNING' if gini < 0.3 else 'FAIL'
    print(f'  {sample}: {gini:.3f} [{status}]')
```

## Read Count Distribution

```python
fig, axes = plt.subplots(1, 2, figsize=(12, 5))

# Log read count distribution
for sample in count_matrix.columns:
    log_counts = np.log10(count_matrix[sample] + 1)
    axes[0].hist(log_counts, bins=50, alpha=0.5, label=sample)
axes[0].set_xlabel('Log10(counts + 1)')
axes[0].set_ylabel('sgRNAs')
axes[0].set_title('Read Count Distribution')
axes[0].legend()

# Cumulative distribution
for sample in count_matrix.columns:
    sorted_counts = np.sort(count_matrix[sample])[::-1]
    cumsum = np.cumsum(sorted_counts) / sorted_counts.sum()
    axes[1].plot(np.arange(len(cumsum)) / len(cumsum) * 100, cumsum * 100, label=sample)
axes[1].set_xlabel('% of sgRNAs (ranked)')
axes[1].set_ylabel('% of total reads')
axes[1].set_title('Cumulative Read Distribution')
axes[1].legend()

plt.tight_layout()
plt.savefig('qc_distribution.png', dpi=150)
```

## Replicate Correlation

**Goal:** Verify that biological and technical replicates are concordant.

**Approach:** Compute pairwise Pearson correlations on log-transformed counts, display as a heatmap, and flag replicate pairs with correlation below 0.8.

```python
# Correlation matrix
log_counts = np.log10(count_matrix + 1)
corr_matrix = log_counts.corr()

plt.figure(figsize=(8, 6))
sns.heatmap(corr_matrix, annot=True, cmap='RdYlBu_r', vmin=0.5, vmax=1,
            square=True, fmt='.2f')
plt.title('Replicate Correlation (log10 counts)')
plt.tight_layout()
plt.savefig('qc_correlation.png', dpi=150)

# Check replicate pairs
print('\nReplicate correlations:')
for i, col1 in enumerate(count_matrix.columns):
    for col2 in count_matrix.columns[i+1:]:
        r = corr_matrix.loc[col1, col2]
        status = 'OK' if r > 0.8 else 'WARNING' if r > 0.6 else 'FAIL'
        print(f'  {col1} vs {col2}: r={r:.3f} [{status}]')
```

## Essential Gene Recovery

**Goal:** Confirm that the screen detects known essential genes as a positive control for screen quality.

**Approach:** Load reference essential gene sets, score genes by MAGeCK negative-selection rank, and compute the AUROC for separating essential from non-essential genes.

```python
# Load known essential genes (e.g., from Hart et al. or DepMap)
essential_genes = set(pd.read_csv('essential_genes.txt', header=None)[0])
nonessential_genes = set(pd.read_csv('nonessential_genes.txt', header=None)[0])

# Load MAGeCK results
results = pd.read_csv('screen.gene_summary.txt', sep='\t')

# Check recovery in T0 vs later timepoint
present_essential = results[results['id'].isin(essential_genes)]
present_nonessential = results[results['id'].isin(nonessential_genes)]

# ROC-like analysis
from sklearn.metrics import roc_auc_score

y_true = results['id'].isin(essential_genes).astype(int)
y_score = -results['neg|score']  # More negative = more essential

if y_true.sum() > 0:
    auc = roc_auc_score(y_true, y_score)
    print(f'\nEssential gene recovery AUC: {auc:.3f}')
    status = 'EXCELLENT' if auc > 0.9 else 'GOOD' if auc > 0.8 else 'FAIR' if auc > 0.7 else 'POOR'
    print(f'Status: {status}')
```

## sgRNA Performance

```python
# sgRNAs per gene
sgrnas_per_gene = genes.value_counts()
print(f'\nsgRNAs per gene: mean={sgrnas_per_gene.mean():.1f}, min={sgrnas_per_gene.min()}, max={sgrnas_per_gene.max()}')

# Check for genes with few sgRNAs
few_sgrnas = sgrnas_per_gene[sgrnas_per_gene < 3]
if len(few_sgrnas) > 0:
    print(f'WARNING: {len(few_sgrnas)} genes have <3 sgRNAs')
```

## Sample Normalization Check

```python
# Total reads per sample
total_reads = count_matrix.sum()
print('\nTotal reads per sample:')
for sample, total in total_reads.items():
    print(f'  {sample}: {total:,}')

# Check for major imbalances
cv = total_reads.std() / total_reads.mean()
print(f'\nCoefficient of variation: {cv:.3f}')
if cv > 0.5:
    print('WARNING: Large variation in sequencing depth')
```

## QC Summary Report

**Goal:** Generate a comprehensive pass/fail QC summary for a CRISPR screen.

**Approach:** Aggregate zero-count percentage, mean Gini index, and minimum replicate correlation into a single report, applying quality thresholds to determine overall screen status.

```python
def generate_qc_report(count_matrix, genes):
    report = {
        'total_sgrnas': len(count_matrix),
        'total_genes': genes.nunique(),
        'samples': len(count_matrix.columns),
        'zero_count_pct': (count_matrix == 0).sum().mean() / len(count_matrix) * 100,
        'gini_mean': count_matrix.apply(gini_index).mean(),
        'replicate_corr_min': np.log10(count_matrix + 1).corr().min().min(),
    }

print('=== QC Summary ===')
    for key, value in report.items():
        if isinstance(value, float):
            print(f'{key}: {value:.3f}')
        else:
            print(f'{key}: {value}')

# Overall status
    passes = []
    passes.append(report['zero_count_pct'] < 5)
    passes.append(report['gini_mean'] < 0.25)
    passes.append(report['replicate_corr_min'] > 0.7)

status = 'PASS' if all(passes) else 'FAIL'
    print(f'\nOverall QC: {status}')

return report

report = generate_qc_report(count_matrix, genes)
```

## Related Skills

- mageck-analysis - Run MAGeCK after QC
- hit-calling - Downstream analysis
- read-qc/quality-reports - General NGS QC