bio-copy-number-gatk-cnv

name	bio-copy-number-gatk-cnv
description	Call copy number variants with the GATK best-practices workflows — the somatic CNV pipeline (CollectReadCounts, DenoiseReadCounts with tangent normalization, ModelSegments, CallCopyRatioSegments) and the germline GATK-gCNV pipeline (DetermineGermlineContigPloidy, GermlineCNVCaller cohort/case mode, PostprocessGermlineCNVCalls). Covers panel-of-normals construction, AnnotateIntervals/FilterIntervals, allelic-count integration, and QS-based filtering. Use when integrating CNV calling into a GATK variant pipeline, calling rare germline CNVs from an exome cohort, deciding between the somatic and germline GATK workflows, or diagnosing why tangent normalization removed a real event or why gCNV output has low precision.
tool_type	cli
primary_tool	gatk

Version Compatibility

Reference examples tested with: GATK 4.5+ (gatk4), Python 3.10+ (gcnv conda env), R 4.3+.

Before using code patterns, verify installed versions match. If versions differ:

• CLI: gatk --version then gatk <ToolName> --help to confirm arguments
• gCNV requires a working gatkcondaenv (theano/tensorflow stack) — gatk will report if the Python environment is missing

GATK 4.5+ gCNV inference defaults are tuned for whole-exome data; whole-genome runs generally need parameter changes. If a tool reports an unrecognized argument, check the help for that exact GATK version rather than retrying.

GATK CNV Workflows

"Call CNVs the GATK way" -> GATK has two separate CNV workflows that share almost no tools. Picking the wrong one is the most common mistake.

• Somatic CNV: CollectReadCounts -> DenoiseReadCounts -> ModelSegments -> CallCopyRatioSegments. Tumor copy-ratio segments, optionally allele-aware.
• Germline gCNV: DetermineGermlineContigPloidy -> GermlineCNVCaller -> PostprocessGermlineCNVCalls. Per-sample germline CN genotypes (VCF).

Critical: What GATK Somatic CNV Does NOT Provide

ModelSegments + CallCopyRatioSegments produce copy-ratio segments and a minor-allele fraction per segment, and the "call" is a simple t-test emitting + / - / 0. This is not integer allele-specific copy number, not tumor purity, and not ploidy. Practitioners routinely assume parity with ASCAT/FACETS and there is none. For integer allele-specific CN, purity, ploidy, LOH state, or whole-genome-doubling status, use allele-specific-copy-number (ASCAT, Sequenza, FACETS, or PureCN — PureCN can even reuse the GATK ModelSegments segmentation as input).

Somatic vs Germline — Choosing the Workflow

Question	Somatic CNV	Germline gCNV
Input	One tumor (+ optional matched normal)	A cohort of constitutional samples
Output	Copy-ratio segments, +/-/0 call, minor-allele fraction	Integer germline CN genotype VCF per sample
Normalization	Tangent (projection onto PoN subspace)	PCA batching + Bayesian read-depth model
Cohort needed	PoN of normals for denoising	>= ~100 technically matched samples (cohort mode)
Use for	Tumor SCNAs, focal amplifications/deletions	Rare/de novo germline CNVs, NDD/Mendelian cohorts

Decision Tree by Scenario

Scenario	Workflow	Key parameters
Tumor-normal WGS/WES, want SCNAs	Somatic, with matched-normal allelic counts	PreprocessIntervals --bin-length 1000 (WGS) or 0 (WES)
Tumor-only somatic CNV	Somatic, no matched-normal allelic counts	Genotype hets in the case sample; expect more no-calls
Rare germline CNV, exome cohort >= 100	gCNV cohort mode	Run DetermineGermlineContigPloidy cohort first
New sample vs an existing gCNV model	gCNV case mode	Must reuse identical scatter count and interval list
Need integer ASCN / purity / ploidy	Neither — escalate	Use allele-specific-copy-number
Targeted panel (< few hundred genes)	Prefer CNVkit	GATK interval models are unstable on tiny panels

Somatic CNV Pipeline

# 1. Preprocess and annotate intervals (WES: bin-length 0 = use exome targets as-is)
gatk PreprocessIntervals -R ref.fa -L targets.interval_list \
    --bin-length 0 --interval-merging-rule OVERLAPPING_ONLY -O preprocessed.interval_list
gatk AnnotateIntervals -R ref.fa -L preprocessed.interval_list \
    --interval-merging-rule OVERLAPPING_ONLY -O annotated.tsv     # GC content for FilterIntervals

# 2. Collect read counts (each BAM)
gatk CollectReadCounts -R ref.fa -I sample.bam -L preprocessed.interval_list \
    --interval-merging-rule OVERLAPPING_ONLY -O sample.counts.hdf5

# 3. Build the panel of normals (tangent-normalization basis)
# --minimum-interval-median-percentile 5.0 is the GATK CNV tutorial value (tool default 10.0)
gatk CreateReadCountPanelOfNormals \
    -I normal1.counts.hdf5 -I normal2.counts.hdf5 -I normalN.counts.hdf5 \
    --annotated-intervals annotated.tsv \
    --minimum-interval-median-percentile 5.0 -O cnv.pon.hdf5

# 4. Denoise tumor against the PoN (tangent normalization)
gatk DenoiseReadCounts -I tumor.counts.hdf5 --count-panel-of-normals cnv.pon.hdf5 \
    --standardized-copy-ratios tumor.standardizedCR.tsv \
    --denoised-copy-ratios tumor.denoisedCR.tsv

# 5. Allelic counts at common biallelic SNPs (tumor and matched normal)
gatk CollectAllelicCounts -R ref.fa -I tumor.bam -L common_snps.interval_list \
    -O tumor.allelicCounts.tsv
gatk CollectAllelicCounts -R ref.fa -I normal.bam -L common_snps.interval_list \
    -O normal.allelicCounts.tsv

# 6. Joint segmentation of copy ratio and allele fraction
gatk ModelSegments --denoised-copy-ratios tumor.denoisedCR.tsv \
    --allelic-counts tumor.allelicCounts.tsv \
    --normal-allelic-counts normal.allelicCounts.tsv \
    --output-prefix tumor -O segments/

# 7. Call each segment +/-/0 (simple t-test against the copy-ratio baseline)
gatk CallCopyRatioSegments -I segments/tumor.cr.seg -O segments/tumor.called.seg

AnnotateIntervals (step 1) and supplying --annotated-intervals to the PoN are frequently skipped — they enable explicit GC-bias correction and are recommended.

Germline gCNV Pipeline

# 1. Determine contig ploidy across the cohort (karyotype + global depth)
gatk DetermineGermlineContigPloidy -L preprocessed.interval_list \
    --interval-merging-rule OVERLAPPING_ONLY \
    -I sample1.counts.hdf5 -I sampleN.counts.hdf5 \
    --contig-ploidy-priors ploidy_priors.tsv --output-prefix cohort -O ploidy-calls/

# 2. FilterIntervals — remove low-mappability / extreme-GC / low-count intervals
gatk FilterIntervals -L preprocessed.interval_list --annotated-intervals annotated.tsv \
    -I sample1.counts.hdf5 -I sampleN.counts.hdf5 \
    --interval-merging-rule OVERLAPPING_ONLY -O filtered.interval_list

# 3. GermlineCNVCaller, cohort mode (builds the model AND calls the cohort)
gatk GermlineCNVCaller --run-mode COHORT -L filtered.interval_list \
    --interval-merging-rule OVERLAPPING_ONLY \
    --contig-ploidy-calls ploidy-calls/cohort-calls \
    -I sample1.counts.hdf5 -I sampleN.counts.hdf5 \
    --output-prefix cohort -O gcnv-calls/

# 4. Post-process per sample into a genotyped VCF
gatk PostprocessGermlineCNVCalls \
    --calls-shard-path gcnv-calls/cohort-calls \
    --model-shard-path gcnv-calls/cohort-model \
    --contig-ploidy-calls ploidy-calls/cohort-calls \
    --sample-index 0 \
    --output-genotyped-intervals sample0.intervals.vcf.gz \
    --output-genotyped-segments sample0.segments.vcf.gz \
    --output-denoised-copy-ratios sample0.denoisedCR.tsv

Case mode (--run-mode CASE) scores a new sample against the cohort *-model shards; it must use the identical filtered.interval_list and the same scatter count as the cohort run, or it fails or produces incomparable calls.

Failure Modes

Tangent normalization removes a real CNV

Trigger: PoN is small (< ~20 normals) or contains samples that share a recurrent CNV (e.g. a common germline CNV, or a PoN accidentally built from tumors).

Mechanism: DenoiseReadCounts projects the tumor coverage profile onto the subspace spanned by the PoN's principal components. Any copy-number pattern present in that subspace is treated as "systematic noise" and subtracted. A CNV shared by PoN members is therefore normalized out of the tumor.

Symptom: A known event (recurrent amplification/deletion, or a common germline CNV) is absent from denoisedCR.tsv; denoised profile is suspiciously flat at that locus.

Fix: Build the PoN from >= 20-40 unrelated, tumor-free, process-matched normals. Never put tumors in the PoN. Cross-check against the standardizedCR.tsv (pre-tangent) profile — if the event is there but gone after denoising, the PoN ate it.

Mistaking ModelSegments output for allele-specific integer CN

Trigger: Treating tumor.modelFinal.seg minor-allele fraction as integer minor copy number, or expecting a purity/ploidy field.

Mechanism: GATK somatic CNV models copy ratio and allele fraction but never fits the purity/ploidy grid that converts log-ratio to integer absolute CN.

Symptom: No purity/ploidy in any output; "copy number" is continuous log2; LOH is a low minor-allele fraction, not an explicit CN-LOH state.

Fix: Accept GATK somatic CNV as a relative caller. For integer ASCN, feed the data to PureCN (segmentationGATK4 reuses GATK segments), FACETS, ASCAT, or Sequenza — see allele-specific-copy-number.

FilterIntervals silently drops intervals containing real variants

Trigger: Aggressive mappability or segmental-duplication cutoffs in FilterIntervals.

Mechanism: A minimum mappability > 0 or a maximum segmental-duplication content < 1 excludes intervals overlapping segdups and low-mappability regions — exactly where many disease-relevant CNVs (e.g. recurrent genomic-disorder loci flanked by segdups) live.

Symptom: Known recurrent CNVs at segdup-mediated loci are never called; the gene of interest has no intervals in filtered.interval_list.

Fix: Inspect filtered.interval_list for genes of interest before calling. Relax mappability/segdup cutoffs for targeted analyses; for genomic-disorder loci, depth-based callers are inherently limited near segdups — confirm with an orthogonal assay.

Raw gCNV output has ~22% precision

Trigger: Using unfiltered GermlineCNVCaller / PostprocessGermlineCNVCalls output for association or de novo analysis.

Mechanism: gCNV is tuned for high recall (~95% of rare coding CNVs >= 2 exons) at the cost of precision; raw calls are dominated by false positives.

Symptom: Implausibly many rare CNVs per sample; de novo CNV rate far above the expected ~0.01-0.02/genome.

Fix: Apply the QS (quality score) filter. QS > 100 is a common starting threshold; QS > 1000 reaches ~96% precision. Also apply sample-level filters (call rate, number of CNVs per sample) per Babadi 2023.

gCNV cohort too small or mismatched

Trigger: Cohort mode with < ~100 samples, or a cohort spanning multiple capture kits / library protocols.

Mechanism: The Bayesian model needs enough technically similar samples to learn coverage bias; mixed protocols are not separable and the model misattributes batch effects to copy number.

Symptom: Unstable calls; many CNVs tracking sequencing batch; model fails to converge.

Fix: Use >= 100 process-matched samples per cohort model; split heterogeneous cohorts by capture kit. For < 100 samples, gCNV case mode against an external compatible model, or a cohort caller like ExomeDepth, is more appropriate — see germline-cnv-interpretation.

Reconciliation: GATK vs Other Callers

Pattern	Likely cause	Action
GATK somatic flat where CNVkit calls a focal event	Tangent normalization absorbed it	Check standardizedCR.tsv; rebuild PoN without the event
GATK and ASCAT disagree on a "deletion"	GATK has no purity model; the event is subclonal or impure	Trust ASCAT/FACETS integer ASCN
gCNV calls a CNV ExomeDepth misses	Different sensitivity profiles; both have poor inter-tool concordance	Require QS filtering + a second caller for rare-CNV claims
gCNV CNV count tracks batch	Cohort mixes protocols	Re-batch by capture kit

Operational rule: GATK somatic CNV output is relative copy ratio — report it as gain/loss/neutral, not absolute CN, unless downstream-fit by an allele-specific tool. gCNV calls are reportable only after QS and sample-level filtering, and rare-CNV or de novo claims need orthogonal confirmation.

Quantitative Thresholds

Threshold	Value	Source / Rationale
PoN size (somatic)	>= 20-40 normals	Larger PoN = stabler tangent subspace; small PoN over-fits
gCNV cohort size	>= ~100 technically matched	Babadi 2023 Nat Genet; model needs coverage-bias signal
gCNV QS for high precision	QS > 1000 -> ~96% precision	Babadi 2023; raw output ~22% precision, ~95% recall
minimum-interval-median-percentile	10.0 default; 5.0 in the GATK CNV tutorial	Drops the lowest-coverage intervals from the PoN
WGS bin length	~1000 bp	PreprocessIntervals --bin-length 1000; WES uses 0 (targets as-is)
Het sites for somatic ModelSegments	>= ~10,000 (WGS)	Sparse hets give noisy minor-allele-fraction segmentation

Common Errors

Error / symptom	Cause	Solution
gatkcondaenv / theano error in gCNV	gCNV Python env not installed	Install the GATK conda env; gCNV cannot run without it
"At least one interval must remain" in FilterIntervals	All intervals filtered out	Relax mappability/GC/count cutoffs; check annotated.tsv
Case-mode gCNV fails or gives odd calls	Different scatter count or interval list vs cohort model	Reuse the exact cohort scatter count and filtered.interval_list
Denoised profile flat at a known event	Tangent normalization removed it	Rebuild PoN larger, tumor-free; inspect standardizedCR
ModelSegments has few het sites	SNP interval list misses captured regions	Use a common-SNP list intersected with the capture targets
Expecting purity/ploidy in output	Somatic CNV does not estimate them	Use allele-specific-copy-number

References

• GATK Best Practices: Somatic copy number variant discovery (CNV). Broad Institute documentation.
• Babadi M et al 2023. GATK-gCNV enables the discovery of rare copy number variants from exome sequencing data. Nat Genet 55:1589
• Gao GF, Oh S, Saksena G, Tabak B, Beroukhim R, Getz G et al 2022. Tangent normalization for somatic copy-number inference in cancer genome analysis. Bioinformatics 38:4677 (Tabak is a middle, not first, author).

Related Skills

• copy-number/allele-specific-copy-number - Integer ASCN, purity, ploidy (ASCAT/Sequenza/FACETS/PureCN)
• copy-number/copy-ratio-segmentation - Segmentation algorithms and depth normalization theory
• copy-number/cnvkit-analysis - Read-depth CNV calling for panels and exomes
• copy-number/germline-cnv-interpretation - ACMG/ClinGen classification of germline CNV calls
• copy-number/cnv-visualization - Plotting GATK denoised ratios and modeled segments
• copy-number/recurrent-cnv - Cohort-level recurrent and driver CNV
• variant-calling/gatk-variant-calling - GATK SNV/indel pipeline for allelic-count SNP sites