Bio Copy Number Cnv Annotation

Version Compatibility

Reference examples tested with: bedtools 2.31+, AnnotSV 3.4+, Python 3.10+ with

pybedtools 0.9+, pandas 2.2+, pysam 0.22+; R 4.3+ with clusterProfiler 4.10+.

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

• CLI: bedtools --version, AnnotSV --version
• Python: pip show pybedtools pandas pysam
• R: packageVersion('clusterProfiler')

If code throws an error, introspect the installed package and adapt the example. AnnotSV

output column names change between major versions — verify against the installed version.

CNV Annotation

"Annotate my CNV calls with the genes they affect" -> Overlap CNV segments with gene models, dosage-sensitivity maps, and clinical databases. The hard part is not the intersection — it is deciding *which* genes matter. A focal amplification overlapping 30 genes usually has one driver (the peak gene); a deletion's consequence depends on whether each gene is dosage-sensitive and whether the whole gene or only part is removed.

• CLI: bedtools intersect -a cnvs.bed -b genes.bed -wa -wb; AnnotSV for full annotation
• Python: pybedtools for interval logic; pysam for VCF database queries

Annotation Strategy — Pick the Database for the Question

| Question | Resource | What it answers |

|----------|----------|-----------------|

| Which genes does this CNV span? | RefSeq/GENCODE gene BED | Raw overlap (not yet consequence) |

| Is loss of this gene damaging? | ClinGen haploinsufficiency (HI) score | Dosage sensitivity to deletion |

| Is gain of this gene damaging? | ClinGen triplosensitivity (TS) score | Dosage sensitivity to duplication |

| Is this CNV common in the population? | gnomAD-SV, DGV, 1000G CNV | Benign-frequency filtering |

| Is this a known recurrent disorder locus? | ClinGen dosage regions, DECIPHER | Genomic-disorder context |

| Is this a cancer driver? | COSMIC Cancer Gene Census, OncoKB | Oncogene vs tumor-suppressor role |

| Is there pathogenic small-variant content? | ClinVar | Coincident SNV/indel pathogenicity |

| One-shot comprehensive annotation + ranking | AnnotSV | Aggregates most of the above |

For constitutional CNV *classification* (assigning pathogenic/VUS/benign), the annotated

output feeds the ACMG/ClinGen points framework — see germline-cnv-interpretation. For

cohort-level recurrence and driver-peak identification, see recurrent-cnv.

The Core Distinction: Overlap Is Not Consequence

A CNV overlapping a gene does not necessarily change that gene's dosage in a way that

matters. Three refinements separate annotation from interpretation:

• Whole-gene vs partial overlap. A deletion spanning an entire gene removes one copy

(clean haploinsufficiency test). A deletion removing only the last two exons creates a

truncated allele — a different, often more damaging, consequence. Always record the

fraction of each gene covered and whether coding exons or only introns/UTRs are hit.

• Dosage sensitivity. Most genes tolerate single-copy loss. ClinGen HI/TS scores

(3 = sufficient evidence for dosage sensitivity, 0 = no evidence, 30 = gene associated

with an autosomal-recessive phenotype, 40 = dosage sensitivity unlikely) indicate which

genes' loss/gain is actually consequential.

• Driver vs passenger in focal events. A focal amplification carries many genes; the

driver is the one under selection, typically at the recurrence peak across a cohort

(GISTIC) and a known oncogene. Annotating all 30 genes as "amplified" overstates.

Gene Overlap with bedtools

Goal: Find genes overlapping each CNV segment, recording overlap extent.

Approach: Convert segments to BED, intersect with a gene model, keep both feature

sets (-wo reports the overlap length) so partial vs whole-gene overlap is recoverable.

# Segments to BED (CNVkit .cns example; columns chrom/start/end/log2)
awk 'NR>1 {print $1"\t"$2"\t"$3"\t"$5}' sample.cns > sample.cnv.bed

# Intersect; -wo appends the number of overlapping bases
bedtools intersect -a sample.cnv.bed -b gencode.genes.bed -wo > cnv_gene_overlap.txt

Comprehensive Annotation with AnnotSV

Goal: Annotate CNVs against genes, dosage maps, population frequency, and clinical

databases in one pass, with a built-in pathogenicity ranking.

Approach: Export CNVs to VCF or BED and run AnnotSV; it returns a "full" line per

gene plus a "split" summary, with an ACMG-aligned rank (1-5).

AnnotSV \
    -SVinputFile sample.cnv.vcf \
    -genomeBuild GRCh38 \
    -annotationMode both \
    -outputFile sample_annotated.tsv

# Output includes: overlapped genes, ClinGen HI/TS, gnomAD-SV/DGV frequency, OMIM,
# ClinVar, DECIPHER, and an ACMG-class rank per SV.

AnnotSV's rank is a useful triage signal, not a final classification — confirm against

the ClinGen points framework for clinical reporting.

Dosage-Sensitivity and Driver Annotation

Goal: Tag each affected gene with its dosage sensitivity and, for tumors, its driver

role, so passengers can be separated from drivers.

Approach: Join the gene-overlap table to the ClinGen dosage map (HI/TS scores) and to

the COSMIC Cancer Gene Census; flag CNVs whose direction matches a known mechanism

(oncogene amplified, tumor suppressor deleted).

import pandas as pd

def annotate_dosage_and_drivers(overlap_tsv, clingen_dosage, cgc_file):
    '''Tag overlapped genes with ClinGen HI/TS and COSMIC driver role.'''
    cols = ['cnv_chrom', 'cnv_start', 'cnv_end', 'log2',
            'gene_chrom', 'gene_start', 'gene_end', 'gene', 'overlap_bp']
    df = pd.read_csv(overlap_tsv, sep='\t', names=cols)
    df['gene_len'] = df['gene_end'] - df['gene_start']
    df['gene_frac_covered'] = (df['overlap_bp'] / df['gene_len']).clip(upper=1.0)
    df['whole_gene'] = df['gene_frac_covered'] >= 0.99

    dosage = pd.read_csv(clingen_dosage, sep='\t')  # gene, HI_score, TS_score
    df = df.merge(dosage, on='gene', how='left')

    cgc = pd.read_csv(cgc_file, sep='\t')
    role = dict(zip(cgc['Gene Symbol'], cgc['Role in Cancer']))
    df['driver_role'] = df['gene'].map(role)

    # Direction-consistent driver hits: oncogene amplified or TSG deleted.
    df['driver_hit'] = (
        ((df['log2'] > 0.3) & df['driver_role'].fillna('').str.contains('oncogene')) |
        ((df['log2'] < -0.3) & df['driver_role'].fillna('').str.contains('TSG')))
    return df

Population-Frequency Filtering

Goal: Remove common, presumed-benign CNVs before clinical interpretation.

Approach: Reciprocal-overlap match each CNV against a population SV catalog

(gnomAD-SV, DGV); a CNV with high reciprocal overlap to a common population CNV of the

same type is likely benign.

# 50% reciprocal overlap (-f 0.5 -r): same-type, similar-extent population match.
# Reciprocal overlap, not one-sided, prevents a tiny CNV inside a huge population CNV
# (or vice versa) from being wrongly matched.
bedtools intersect -a sample.cnv.bed -b gnomad_sv.bed -f 0.5 -r -wa -wb \
    > cnv_population_match.txt

Pathway Enrichment of Affected Genes

Goal: Test whether genes in amplified (or deleted) regions are enriched for pathways.

Approach: Extract genes by CNV direction, map to Entrez IDs, run GO/KEGG enrichment.

Caveat: CNVs are large and gene-dense, so enrichment is biased toward whatever pathways

cluster in CNV-prone genomic regions — interpret as hypothesis-generating.

library(clusterProfiler)
library(org.Hs.eg.db)

amp_genes <- unique(cnv_annot$gene[cnv_annot$log2 > 0.3])
entrez <- na.omit(mapIds(org.Hs.eg.db, keys = amp_genes,
                         keytype = 'SYMBOL', column = 'ENTREZID'))
go_bp <- enrichGO(gene = entrez, OrgDb = org.Hs.eg.db, ont = 'BP',
                  pAdjustMethod = 'BH', qvalueCutoff = 0.05)

Failure Modes

Genome-build mismatch between CNVs and annotation

Trigger: CNV coordinates on GRCh37 intersected with a GRCh38 gene model (or vice versa).

Mechanism: Coordinates silently shift; the intersection succeeds and returns wrong genes.

Symptom: Implausible gene assignments; a known driver locus annotated with the wrong gene; systematic offset.

Fix: Confirm both inputs are the same build. If not, liftOver the CNVs (note that liftOver can split or drop segments across assembly gaps) and verify a known landmark.

Annotating all overlapped genes as the "affected" genes

Trigger: Reporting every gene a focal amplification spans as amplified/driver.

Mechanism: Focal events are megabases wide and gene-dense; only the selected gene is the driver.

Symptom: A 2 Mb amplicon "amplifies" 40 genes; the report cannot distinguish ERBB2 from its passengers.

Fix: For focal events, prioritize the gene at the cohort recurrence peak (GISTIC, see recurrent-cnv) and known drivers (CGC/OncoKB). Report passengers separately or not at all.

ClinVar CLNSIG parsing errors

Trigger: Naive string matching on the ClinVar CLNSIG INFO field.

Mechanism: CLNSIG is multi-valued, mixes terms ("Conflicting_classifications", "Pathogenic/Likely_pathogenic", "Benign/Likely_benign"), and is per-small-variant — not per-CNV. A substring match for "pathogenic" silently captures "Likely_pathogenic" (intended) but a careless match also fires on records that are conflicting or benign once underscores and slashes are involved.

Symptom: Benign or conflicting variants reported as pathogenic; CNV flagged on incidental nearby SNVs.

Fix: Parse CLNSIG against the controlled vocabulary; exclude "Conflicting" and benign terms explicitly. Remember ClinVar SNV/indel pathogenicity does not transfer to a CNV — use it as context, and use ClinVar's own CNV records or ClinGen dosage regions for the CNV itself.

Equating overlap with consequence

Trigger: Treating any gene-overlapping CNV as functionally significant.

Mechanism: Most single-copy losses are tolerated; partial overlaps may hit only introns/UTRs.

Symptom: Long lists of "affected" dosage-insensitive genes; benign CNVs over-called as significant.

Fix: Require dosage evidence (ClinGen HI/TS) and record coding-exon overlap and whole-gene-vs-partial status before calling a gene affected.

Quantitative Thresholds

| Threshold | Value | Source / Rationale |

|-----------|-------|--------------------|

| Population-CNV reciprocal overlap | >= 50% (-f 0.5 -r) | Standard reciprocal-overlap match for benign filtering (convention traces to gnomAD-SV / DGV workflows; Collins SR et al 2020 *Nature* 581:444 uses comparable reciprocal-overlap thresholds for benign-population matching) |

| Common-CNV benign frequency | > 1% population frequency | ACMG/ClinGen: high frequency supports benign |

| ClinGen HI/TS dosage-sensitive | score = 3 | ClinGen: sufficient evidence for dosage sensitivity |

| Whole-gene overlap | >= 99% gene length covered | Distinguishes clean haploinsufficiency from partial/truncating |

| AnnotSV pathogenic ranks | rank 4-5 | AnnotSV ACMG-aligned ranking (1 benign - 5 pathogenic) |

Common Errors

| Error / symptom | Cause | Solution |

|-----------------|-------|----------|

| Wrong genes assigned to a CNV | hg19/hg38 build mismatch | Match builds; liftOver and verify a landmark |

| 40 genes called "amplified" for one amplicon | All overlapped genes reported as drivers | Prioritize recurrence-peak + known-driver genes |

| Benign variants flagged pathogenic | Substring match on ClinVar CLNSIG | Parse the controlled vocabulary explicitly |

| Tiny CNV matched to a huge population CNV | One-sided overlap used for frequency filter | Use reciprocal overlap (-f 0.5 -r) |

| AnnotSV columns not found | Column names differ across AnnotSV versions | Check head of the output for the installed version |

| Enrichment dominated by gene-dense loci | CNVs span gene clusters | Treat CNV-gene enrichment as hypothesis-generating |

References

• Geoffroy V et al 2018. AnnotSV: an integrated tool for structural variations annotation. Bioinformatics 34:3572
• Riggs ER et al 2020. Technical standards for the interpretation and reporting of constitutional copy-number variants: ACMG and ClinGen. Genet Med 22:245
• Collins RL et al 2020. A structural variation reference for medical and population genetics (gnomAD-SV). Nature 581:444
• Sondka Z et al 2018. The COSMIC Cancer Gene Census. Nat Rev Cancer 18:696

Related Skills

• copy-number/germline-cnv-interpretation - ACMG/ClinGen points-based CNV classification
• copy-number/recurrent-cnv - GISTIC2 recurrence peaks and driver-gene identification
• copy-number/cnvkit-analysis - Generates the CNV segments to annotate
• copy-number/cnv-visualization - Visualizing annotated CNVs
• pathway-analysis/go-enrichment - GO/KEGG enrichment methodology and caveats
• genome-intervals/bed-file-basics - BED interval operations
• clinical-databases/clinvar-lookup - Querying ClinVar for variant pathogenicity