Quality control for pooled CRISPR screens covering library representation, Gini index, log-skew, replicate Pearson and Spearman concordance, essentialome precision-recall AUC against CEGv2 (Hart 2017), Cas9 cut-toxicity diagnostics, copy-number amplicon detection (Aguirre 2016 / Munoz 2016), bottleneck propagation through plasmid pool, infection, selection, and endpoint stages, MOI verification, and DepMap-style screen-quality scoring. Use when assessing screen quality before hit calling, deciding whether to repeat or rescue a screen, diagnosing low-confidence hits, choosing between MAGeCK / BAGEL2 / Chronos based on quality grade, picking a normalization strategy from QC signatures, or evaluating whether an in-vivo screen retained adequate library complexity.
Quality control for pooled CRISPR screens covering library representation, Gini index, log-skew, replicate Pearson and Spearman concordance, essentialome precision-recall AUC against CEGv2 (Hart 2017), Cas9 cut-toxicity diagnostics, copy-number amplicon detection (Aguirre 2016 / Munoz 2016), bottleneck propagation through plasmid pool, infection, selection, and endpoint stages, MOI verification, and DepMap-style screen-quality scoring. Use when assessing screen quality before hit calling, deciding whether to repeat or rescue a screen, diagnosing low-confidence hits, choosing between MAGeCK / BAGEL2 / Chronos based on quality grade, picking a normalization strategy from QC signatures, or evaluating whether an in-vivo screen retained adequate library complexity.
Before using code patterns, verify installed versions match. If versions differ:
CLI: mageck --version then mageck count --help; R: packageVersion('MAGeCKFlute')
R: packageVersion('MAGeCKFlute') then ?BatchRemove / ?FluteRRA
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
"Audit my CRISPR screen quality before hit calling" -> Assess library representation, replicate concordance, depth, drift, and biological signal recovery using DepMap-grade metrics, then decide whether the screen is usable, salvageable, or must be repeated.
Python: pandas + scikit-learn for Gini, AUC, PCA; MAGeCKFlute (R) for one-shot QC dashboard
CLI: mageck count writes Gini and mapping stats unconditionally to <prefix>.countsummary.txt (GiniIndex, Reads, Mapped, Percentage); MAGeCK-VISPR for an interactive dashboard
QC Stage Hierarchy
A pooled screen has six distinct bottlenecks where complexity can collapse. Audit each:
Stage
Metric
Acceptable threshold
Failure consequence
Plasmid pool
Gini, skew, % zero-count guides
Gini <0.1, skew <2 (Joung 2017 states <10), zero <0.5%
Missing guides cannot be screened; dropout indistinguishable from non-coverage
No correlation between sgRNA off-target count and depletion
False-positive essentiality at amplicons; ERBB2 in HER2+ etc.
Each metric below quantifies one of these stages.
Library Representation Metrics
Goal: Detect dropout, oversaturation, and library bottlenecks at each sequencing stage.
Approach: Compute per-sample zero-count fraction, low-count fraction (<30 reads, the CRISPRcleanR ccr.NormfoldChanges default), and percentile-based skew, then track how these change between plasmid -> Day-0 -> endpoint to localize the bottleneck.
Interpretation: Plasmid pool failing Gini <0.1 indicates synthesis or amplification bias; the screen is unfit for use. Endpoint Gini drifting above 0.30 indicates either heavy biological selection (acceptable for strong-phenotype drug screens) or a bottleneck (must be diagnosed). The Day-0 vs plasmid delta isolates whether the issue arose during infection (cloning is unlikely to lose specific guides between extraction and infection -- the change happens in cells).
Gini Coefficient
Goal: Quantify how unevenly reads are distributed across sgRNAs in a single sample.
Approach: Sort non-zero counts ascending, compute Gini via the cumulative-fraction formula. Compare against stage-specific thresholds.
Stage-specific thresholds: only the plasmid Gini <=0.1 is a published cutoff (MAGeCK-VISPR); the remaining grades are operational convention.
Stage
Excellent
Acceptable
Concerning
Failure
Plasmid pool
<0.10
<0.15
0.15-0.20
>0.20
Day 0 (post-infection)
<0.12
<0.18
0.18-0.25
>0.25
Endpoint (post-selection)
<0.30
<0.40
0.40-0.55
>0.55
A Gini that climbs from 0.10 (plasmid) to 0.45 (endpoint) is expected when the screen exerts strong selection (drug, lethal-condition). A Gini that climbs to 0.45 without any biological selection (e.g., a control-vs-control timepoint comparison) indicates technical drift.
Replicate Concordance
Goal: Verify that biological/technical replicates agree before testing for between-condition differences.
Approach: Compute pairwise Pearson on log10(counts+1) (MAGeCK-VISPR convention) and Spearman ρ on raw rank, between every replicate pair within a condition. Flag any pair below the MAGeCK-VISPR floor of 0.8 Pearson on log-scale.
Thresholds: 0.8 Pearson is the MAGeCK-VISPR floor; the stricter grades are operational convention.
Metric
Excellent
Acceptable
Failure
Pearson on log10(counts+1)
>0.95
>0.85
<0.80
Spearman on raw ranks
>0.85
>0.70
<0.60
When Pearson is high but Spearman is low, a few outlier sgRNAs are driving correlation (one extreme guide dominates). Inspect the scatterplot; typically caused by PCR jackpotting at a single guide. Hit calling should use a method that ranks (RRA, drugZ) rather than one that fits per-sgRNA fold change directly.
Essentialome Recovery (CEGv2 PR-AUC)
Goal: Verify the screen has detectable biological essentiality signal by checking whether known essentials (Hart 2017 CEGv2) drop out faster than known non-essentials (NEGv1).
Approach: Compute precision-recall AUC where positives are CEGv2 genes and negatives are NEGv1; the screen "passes" if PR-AUC >0.7 (community convention; the CEGv2/NEGv1 sets come from Hart 2017 / Hart 2014).
Source / threshold: Hart 2017 G3 7:2719 defines CEGv2 (~684 core essentials); NEGv1 (~927 non-essentials) comes from Hart 2014 Mol Syst Biol 10:733. Both lists at https://github.com/hart-lab/bagel/blob/master/CEGv2.txt and NEGv1.txt. DepMap convention: PR-AUC >0.7 at FDR 5% is the "passing" threshold; <0.5 means the screen has no essentiality signal and is not interpretable.
When PR-AUC is low despite good Gini and Pearson: cause is usually one of (a) Cas9 was not selected for before screen start (lots of Cas9-negative cells in the pool diluting signal), (b) puromycin selection truncated too aggressively (over-bottleneck), (c) the timepoint is too early (need 14-21 days for KO + decay + selection to manifest). Each has a different remediation.
Copy-Number Amplicon Bias Diagnostic
Goal: Detect the Aguirre 2016 / Munoz 2016 copy-number artifact where sgRNAs targeting amplified loci appear "essential" purely from DNA-damage burden.
Approach: Bin genes by copy number (if known from matched WGS/SNP-array) and check whether mean LFC correlates with CN. Alternatively, count off-target cut sites per sgRNA and check correlation with depletion -- amplified loci share many identical cut sites.
Interpretation: A Spearman ρ < -0.1 between copy number and LFC indicates copy-number artifact. The diagnostic threshold is conservative -- Aguirre 2016 showed the effect scales with copy number and with the number of cut sites per sgRNA. Remediation: use CRISPRcleanR, CERES, or Chronos (see [[copy-number-correction]]) before hit calling.
Sequencing Depth Audit
Goal: Verify that sequencing depth is sufficient to resolve fold changes at the smallest interesting effect size.
Approach: Compute reads/sgRNA per sample and the coefficient of variation (CV) of total reads across samples. Compare against Joung 2017's >100 reads/sgRNA for plasmid QC and >500 for screening, or MAGeCK-VISPR's 300x.
CV interpretation: CV >0.5 across samples in total reads indicates demultiplexing imbalance or library-pooling error; even if individual samples pass depth thresholds, the relative count is then biased.
MOI Verification
Goal: Confirm that infection occurred at MOI 0.3-0.5 so that ≤1 sgRNA/cell predominates.
Approach: From titration plate (control wells with serial-diluted virus), compute infection efficiency, then verify by qPCR of integrated proviral copy number in the screen pool.
MOI
P(≥1 sgRNA/cell)
P(≥2 sgRNAs/cell)
Cells with 2+ guides as fraction of infected
0.3
26%
4%
14%
0.5
39%
9%
23%
1.0
63%
26%
41%
Decision rule: Always titrate to 0.3. At 0.5, 14-23% of "perturbed" cells carry combinatorial perturbations that confound single-gene scoring. The Poisson math is non-negotiable -- there is no analytical correction for high-MOI confounding.
PCA and Batch Effect Detection
Goal: Visualize whether samples cluster by biology or by batch.
Approach: PCA on log10(counts+1); samples should cluster by condition, not by batch/replicate-day/library-lot.
from sklearn.decomposition import PCA
defscreen_pca(counts_df, metadata_df, condition_col='condition'):
'''metadata_df: rows = samples, columns include condition_col, batch (optional).'''
log_counts = np.log10(counts_df + 1).T # samples as rows for PCA
pca = PCA(n_components=3)
pcs = pca.fit_transform(log_counts)
out = pd.DataFrame(pcs, columns=['PC1', 'PC2', 'PC3'], index=counts_df.columns)
out = out.join(metadata_df)
return out, pca.explained_variance_ratio_
Interpretation: If PC1 separates batches, see [[batch-correction]]. If PC1 separates conditions cleanly, the screen has interpretable biology. If neither separates anything, the screen has no signal (failed) or is dominated by technical noise.
Composite DepMap-Style Quality Score
Goal: Generate a single quality grade combining all metrics for pipeline gating.
Approach: Rescale each metric to a comparable 0-1 direction and average them into a single gate score. Screens scoring <-1 SD are typically excluded from DepMap.
defcomposite_qc_score(per_sample_qc):
'''per_sample_qc: one row per sample, joining library_representation() output
(n_sgrnas_detected, reads_per_sgrna) with gini, pearson_min_replicate, pr_auc
and n_sgrnas_total.'''
metrics = {
'gini_inv': 1 - per_sample_qc['gini'],
'pearson': per_sample_qc['pearson_min_replicate'],
'pr_auc': per_sample_qc['pr_auc'],
'depth_log': np.log10(per_sample_qc['reads_per_sgrna']),
'detected_frac': per_sample_qc['n_sgrnas_detected'] / per_sample_qc['n_sgrnas_total'],
}
return pd.DataFrame(metrics).mean(axis=1)
This is a pipeline gate, not a publication metric. DepMap reports gene effect score quality (Chronos-derived) separately from screen quality; Pacini 2021 scores the latter with NNMD.
Failure Modes
High Gini in plasmid pool despite passing all design rules
Trigger: Library was cloned and amplified through too many PCR cycles (>20) or used a high-GC-bias polymerase.
Mechanism: Each PCR cycle compounds GC bias by ~5%; high-GC and low-GC guides become non-linear functions of starting abundance.
Symptom: Gini >0.15 in plasmid, GC-content stratification of dropout.
Fix: Cap PCR at 15 cycles for amplification; use Q5 / NEBNext Ultra II / KAPA HiFi (low-bias); re-sequence post-amp; if still bad, re-clone from glycerol stock.
Falling PR-AUC across timepoints despite stable Gini
Trigger: Cas9 was not selected for before screen start; Cas9-negative cells in the pool dilute essentiality signal.
Mechanism: Each Cas9-negative cell carries a sgRNA but no editing; its sgRNA persists despite biological essentiality of the target.
Symptom: PR-AUC declines from 0.7 at week 1 to 0.4 at week 3; Gini and Pearson both pass.
Fix: Always select Cas9-positive cells (FACS or blast) before infection. For a salvage of an already-run screen, model Cas9-expression heterogeneity as a noise floor and accept reduced sensitivity.
Apparent essentiality of amplified loci
Trigger: Cancer cell line with focal amplification (ERBB2 in SK-BR-3, MYC in colorectal, FGFR1 in head and neck).
Mechanism: Aguirre 2016 / Munoz 2016: many simultaneous Cas9 cuts trigger a DNA-damage response and G2 arrest; sgRNAs at amplified loci appear depleted independently of target essentiality.
Symptom: Hits include genes within known amplicons; sgRNAs with more genome-wide cut sites are more depleted.
Fix: Apply CRISPRcleanR pre-hoc or use Chronos/CERES with matched CN profile (see [[copy-number-correction]]). Always required for cancer-cell-line screens, not optional.
Outlier replicate dragging Pearson down
Trigger: One technical replicate had a library-prep failure (low input, PCR jackpot, sequencing-lane swap).
Mechanism: Outlier sample has different total reads or different per-sgRNA distribution but passes individual sample QC.
Symptom: Pearson between replicates 0.85-0.90 with one pair as outlier; condition-level means look fine.
Fix: Drop the outlier replicate; re-derive Pearson on the remaining pair. If only two replicates and one is outlier, the condition lacks replication and must be re-run.
Low Day-0 coverage from high MOI
Trigger: Infection at MOI >0.5.
Mechanism: Poisson: at MOI 0.5, 23% of infected cells carry multiple sgRNAs; the "single-perturbation" assumption underlying every analysis method is violated.
Symptom: Apparent gene-gene interactions in single-gene screens; gene-level z-scores noisy; Pearson lower than expected for high-quality counts.
Fix: No analytical correction. Re-titrate, re-infect at MOI 0.3, re-run screen.
CRISPRi/a screen with no signal on validated essentials
Trigger: Library targets wrong TSS (Ensembl canonical vs FANTOM5 highest-rank).
Mechanism: dCas9-KRAB knockdown is maximal within ±100 bp of the actual Pol II loading site; canonical annotation can be off 1-10 kb.
Symptom: RPS/RPL/EIF families dropping out as expected (these have clean canonical TSSs) but downstream genes failing; PR-AUC on broader CEGv2 panel drops.
Fix: Re-design library against FANTOM5 highest-CAGE-peak TSS (Sanson 2018); for tissue-specific lines, use matched CAGE / GRO-seq.
Quantitative Thresholds
Threshold
Value
Source / Rationale
Plasmid Gini
<0.10
Li W et al 2015 MAGeCK-VISPR Genome Biol 16:281
Plasmid skew ratio (p90/p10)
<10 (Joung 2017); <2 is a stricter modern convention
Joung 2017 Nat Protoc 12:828
% zero-count sgRNAs (plasmid)
<0.5%
Joung 2017 Nat Protoc 12:828
% zero-count sgRNAs (endpoint)
<1% ideal, <5% tolerated
Li W et al 2015 Genome Biol 16:281
Replicate Pearson on log10(counts+1)
>=0.8 (MAGeCK-VISPR floor); >0.95 ideal
Li W et al 2015 MAGeCK-VISPR Genome Biol 16:281
Replicate Spearman
>0.70
Operational convention
CEGv2 PR-AUC at FDR 5%
>0.70 passing; >0.85 high quality
Community convention (CEGv2 from Hart 2017 G3 7:2719)
Reads per sgRNA per sample
>100 plasmid QC and >500 screening (Joung 2017); 300+ (MAGeCK-VISPR)
Joung 2017; Li W et al 2015
Library coverage at infection
500x cells/sgRNA
Joung 2017; DepMap
In-vivo coverage
50-200x at endpoint
Bottleneck-limited; see [[in-vivo-screens]]
MOI at infection
0.3 strict
Poisson: P(≥2)=4% at 0.3 vs 9% at 0.5
CN-bias Spearman ρ (LFC vs copy number)
abs(ρ) <0.10
Operational convention
Common Errors
Error / symptom
Cause
Solution
Plasmid Gini >0.2
PCR over-amplification
Re-sequence; cap PCR at 15 cycles
Endpoint PR-AUC <0.5
Cas9 not selected pre-screen
Select Cas9+; redo if mid-screen
Pearson high, Spearman low
A few outlier sgRNAs dominate
Use RRA / rank-based hit calling
Replicate Pearson <0.8
Library-prep failure on one rep
Drop outlier; re-run if singleton
% zero increases dramatically Day-0 -> endpoint
Selection bottleneck
Reduce selection pressure; or increase coverage
Top hits include amplified-region genes
CN bias
CRISPRcleanR or Chronos
MOI verification shows 0.6+
Over-infected
Re-run at lower MOI; no rescue
Detected fraction <90% in Day 0
Coverage too low
Increase cells; expect drift
References
Joung J et al. 2017. Nat Protoc 12:828. Genome-wide screen protocol; coverage and depth conventions.
Li W et al. 2014. Genome Biol 15:554. MAGeCK.
Li W et al. 2015. Genome Biol 16:281. MAGeCK-VISPR; Gini, zero-count, depth and replicate-correlation QC cutoffs.
Wang B et al. 2019. Nat Protoc 14:756. MAGeCKFlute; QC dashboard.
Hart T et al. 2017. G3 7:2719. CEGv2 core-essential reference set; PR-AUC screen-quality benchmarking.
Hart T et al. 2014. Mol Syst Biol 10:733. Gold-standard essential and non-essential reference sets; source of NEGv1.
Aguirre AJ et al. 2016. Cancer Discov 6:914. Copy-number amplicon false-essentiality.
Munoz DM et al. 2016. Cancer Discov 6:900. Copy-number gene-independent toxicity.
Pacini C et al. 2021. Nat Commun 12:1661. Integrated cross-study dependencies; NNMD screen-quality metric and cross-study batch correction.
Meyers RM et al. 2017. Nat Genet 49:1779. CERES; mechanism of CN bias.
