variant-annotation

name	variant-annotation
description	Comprehensive variant annotation using bcftools annotate/csq, VEP, SnpEff, and ANNOVAR. Add database annotations, predict functional consequences, and assess clinical significance with MANE transcript selection and pathogenicity scoring. Use when annotating variants with functional and clinical information.
tool_type	mixed
primary_tool	VEP

Version Compatibility

Reference examples tested with: bcftools 1.19+, VEP 110+, SnpEff 5.2+

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

• Python: pip show <package> then help(module.function) to check signatures
• CLI: <tool> --version then <tool> --help to confirm flags
• Note: SnpEff and SnpSift use single-dash -version, not --version

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

Variant Annotation

Tool Comparison

Tool	Best For	Speed	Output
bcftools csq	Simple consequence prediction	Fast	VCF
VEP	Comprehensive with plugins	Moderate	VCF/TXT
SnpEff	Fast batch annotation	Fast	VCF
ANNOVAR	Flexible databases	Moderate	TXT

Normalization Before Annotation

Variant normalization is mandatory before annotation. The same variant represented differently (e.g., left-aligned vs right-aligned indels, multiallelic vs biallelic) produces different annotations. Always normalize first:

bcftools norm -f reference.fa -m-any input.vcf.gz -Oz -o normalized.vcf.gz

The -m-any flag splits multiallelic records into biallelic, ensuring each allele gets its own annotation. Without this step, downstream tools may annotate only the first alternate allele or produce ambiguous consequence calls.

Transcript Selection Strategy

Transcript choice determines which consequence is reported. This single decision changes whether a variant is classified as missense, synonymous, or intronic.

Strategy	When to Use	Recommended?
MANE Select	Clinical reporting; one transcript per gene	Yes -- current standard
MANE Plus Clinical	Genes with clinically relevant non-MANE transcripts	Yes -- supplements MANE Select
Canonical (Ensembl)	Legacy pipelines	Being phased out in favor of MANE
Longest transcript	Legacy convention	No -- may miss clinically relevant variants in shorter isoforms
All transcripts	Research; comprehensive annotation	Good for discovery; report worst consequence per ACMG guidelines

VEP flags: --mane_select adds MANE Select transcript info to output. --pick selects one consequence per variant using VEP's internal priority ranking (canonical > biotype > length). For clinical applications, using both together reports the MANE Select consequence when available.

Tool Concordance

Concordance between VEP, SnpEff, and ANNOVAR is lower than commonly assumed: only 58.52% agreement on HGVSc notation, 84.04% on HGVSp, and 85.58% on coding impact classification. Loss-of-function discrepancies affect ACMG PVS1 interpretation in 33-44% of cases.

Practical implication: for clinical applications, annotate with multiple tools and compare results. For research, pick one tool and apply it consistently across all samples. VEP with MANE Select is the current community standard for clinical genomics.

Clinical vs Research Annotation

Aspect	Clinical	Research
Transcript	MANE Select mandatory	Any consistent approach
Tools	VEP + SnpEff cross-validation recommended	Single tool acceptable
Pathogenicity	ACMG criteria (PP3/BP4 for computational)	Score-based ranking
Reporting	Only clinically significant variants	All variants of interest
Databases	ClinVar (reviewed), HGMD (licensed)	ClinVar, gnomAD, research DBs
Re-analysis	Required periodically (new evidence)	Optional

bcftools annotate

Goal: Add or remove INFO/ID annotations from external databases using bcftools.

Approach: Match variants by position and allele against annotation VCF/BED/TAB files, copying specified columns.

"Add rsIDs to my VCF from dbSNP" -> Match variant positions against a database and copy identifiers or annotation fields into the VCF.

Add Annotations from Database

bcftools annotate -a dbsnp.vcf.gz -c ID input.vcf.gz -Oz -o annotated.vcf.gz

Annotation Columns (-c)

Option	Description
ID	Copy ID column
INFO	Copy all INFO fields
INFO/TAG	Copy specific INFO field
+INFO/TAG	Add to existing values

Add Multiple Annotations

bcftools annotate -a database.vcf.gz -c ID,INFO/AF,INFO/CAF input.vcf.gz -Oz -o annotated.vcf.gz

Add from BED/TAB Files

# BED with 4th column as annotation
bcftools annotate -a regions.bed.gz -c CHROM,FROM,TO,INFO/REGION \
    -h <(echo '##INFO=<ID=REGION,Number=1,Type=String,Description="Region name">') \
    input.vcf.gz -Oz -o annotated.vcf.gz

# Tab file: CHROM POS VALUE
bcftools annotate -a annotations.tab.gz -c CHROM,POS,INFO/SCORE \
    -h <(echo '##INFO=<ID=SCORE,Number=1,Type=Float,Description="Custom score">') \
    input.vcf.gz -Oz -o annotated.vcf.gz

Remove Annotations

bcftools annotate -x INFO/DP,INFO/MQ input.vcf.gz -Oz -o clean.vcf.gz
bcftools annotate -x INFO input.vcf.gz -Oz -o minimal.vcf.gz  # Remove all INFO

Set ID from Fields

bcftools annotate --set-id '%CHROM\_%POS\_%REF\_%ALT' input.vcf.gz -Oz -o with_ids.vcf.gz

bcftools csq

Goal: Predict functional consequences of variants using gene annotations.

Approach: Map variants to GFF3 gene models and classify as synonymous, missense, frameshift, etc.

Simple consequence prediction using GFF annotation.

bcftools csq -f reference.fa -g genes.gff3.gz input.vcf.gz -Oz -o consequences.vcf.gz

Consequence Types

Consequence	Description
synonymous	No amino acid change
missense	Amino acid change
stop_gained	Introduces stop codon
frameshift	Changes reading frame
splice_donor/acceptor	Affects splicing

Ensembl VEP

Goal: Annotate variants comprehensively with consequence, impact, pathogenicity scores, and population frequencies.

Approach: Run VEP with offline cache, enabling SIFT, PolyPhen, HGVS, frequency, and plugin-based predictions.

"Annotate my variants with functional consequences" -> Predict coding effects, impact severity, and pathogenicity using Ensembl's Variant Effect Predictor.

Installation

conda install -c bioconda ensembl-vep
vep_install -a cf -s homo_sapiens -y GRCh38 --CONVERT

Basic Annotation

vep -i input.vcf -o output.vcf --vcf --cache --offline

Comprehensive Annotation

vep -i input.vcf -o output.vcf \
    --vcf \
    --cache --offline \
    --species homo_sapiens \
    --assembly GRCh38 \
    --everything \
    --fork 4

--everything Enables

• --sift b - SIFT predictions
• --polyphen b - PolyPhen predictions
• --hgvs - HGVS nomenclature
• --symbol - Gene symbols
• --canonical - Canonical transcript
• --af - 1000 Genomes frequencies
• --af_gnomade/g - gnomAD frequencies
• --pubmed - PubMed IDs

Filter by Impact

vep -i input.vcf -o output.vcf --vcf \
    --cache --offline \
    --pick \
    --filter "IMPACT in HIGH,MODERATE"

Plugins

# CADD scores
vep -i input.vcf -o output.vcf --vcf \
    --cache --offline \
    --plugin CADD,whole_genome_SNVs.tsv.gz

# dbNSFP (multiple predictors)
vep -i input.vcf -o output.vcf --vcf \
    --cache --offline \
    --plugin dbNSFP,dbNSFP4.3a.gz,ALL

# Multiple plugins
vep -i input.vcf -o output.vcf --vcf \
    --cache --offline \
    --plugin CADD,cadd.tsv.gz \
    --plugin dbNSFP,dbnsfp.gz,SIFT_score,Polyphen2_HDIV_score \
    --plugin SpliceAI,spliceai.vcf.gz

VEP Output Fields

Field	Description
Consequence	SO term (e.g., missense_variant)
IMPACT	HIGH, MODERATE, LOW, MODIFIER
SYMBOL	Gene symbol
HGVSc/HGVSp	HGVS coding/protein change
SIFT/PolyPhen	Pathogenicity predictions

SnpEff

Goal: Annotate variants with gene effects and impact categories using SnpEff.

Approach: Run SnpEff ann against a genome database, then use SnpSift for database cross-referencing and filtering.

Installation

conda install -c bioconda snpeff
snpEff download GRCh38.105

Memory

SnpEff loads the genome database into memory as Java objects. Set -Xmx larger than the database size on disk -- human genome databases (e.g. GRCh38) expand to 3-4 GB in memory and need at least -Xmx8g. Smaller genomes can use less. Without sufficient heap, SnpEff will hit OutOfMemoryError or thrash on garbage collection.

Basic Annotation

snpEff ann GRCh38.105 input.vcf > output.vcf

With Statistics

snpEff ann -v -stats stats.html -csvStats stats.csv GRCh38.105 input.vcf > output.vcf

Filter by Impact

snpEff ann GRCh38.105 input.vcf | \
    SnpSift filter "(ANN[*].IMPACT = 'HIGH')" > high_impact.vcf

SnpEff Impact Categories

Impact	Examples
HIGH	Stop gained, frameshift, splice donor/acceptor
MODERATE	Missense, inframe indel
LOW	Synonymous, splice region
MODIFIER	Intron, intergenic, UTR

SnpSift Database Annotations

# dbSNP
SnpSift annotate dbsnp.vcf.gz input.vcf > annotated.vcf

# ClinVar
SnpSift annotate clinvar.vcf.gz input.vcf > annotated.vcf

# dbNSFP
SnpSift dbnsfp -db dbNSFP4.3a.txt.gz input.vcf > annotated.vcf

# Chain multiple
snpEff ann GRCh38.105 input.vcf | \
    SnpSift annotate dbsnp.vcf.gz | \
    SnpSift annotate clinvar.vcf.gz > fully_annotated.vcf

SnpSift Filtering

SnpSift filter "(QUAL >= 30) & (DP >= 10)" input.vcf > filtered.vcf
SnpSift filter "(exists CLNSIG) & (CLNSIG has 'Pathogenic')" input.vcf > pathogenic.vcf

ANNOVAR

Goal: Annotate variants with gene, frequency, and pathogenicity databases using ANNOVAR.

Approach: Run table_annovar.pl with multiple protocols (gene, filter, region) against downloaded annotation databases.

Installation

# Download from https://annovar.openbioinformatics.org/ (registration required)
annotate_variation.pl -buildver hg38 -downdb -webfrom annovar refGene humandb/
annotate_variation.pl -buildver hg38 -downdb -webfrom annovar gnomad30_genome humandb/

Table Annotation

table_annovar.pl input.vcf humandb/ \
    -buildver hg38 \
    -out annotated \
    -remove \
    -protocol refGene,gnomad30_genome,clinvar_20230416,dbnsfp42a \
    -operation g,f,f,f \
    -nastring . \
    -vcfinput

Python: Parse Annotated VCF

Goal: Extract and interpret annotation fields from VEP CSQ or SnpEff ANN strings in Python.

Approach: Parse pipe-delimited annotation strings against the header-defined field order, then filter by impact or consequence.

Parse VEP CSQ

from cyvcf2 import VCF

def parse_vep_csq(csq_string, csq_header):
    fields = csq_header.split('|')
    values = csq_string.split('|')
    return dict(zip(fields, values))

vcf = VCF('vep_output.vcf')
csq_header = None
for h in vcf.header_iter():
    if h['HeaderType'] == 'INFO' and h['ID'] == 'CSQ':
        csq_header = h['Description'].split('Format: ')[1].rstrip('"')
        break

for variant in vcf:
    csq = variant.INFO.get('CSQ')
    if csq:
        for transcript in csq.split(','):
            parsed = parse_vep_csq(transcript, csq_header)
            if parsed.get('IMPACT') in ('HIGH', 'MODERATE'):
                print(f"{variant.CHROM}:{variant.POS} {parsed['SYMBOL']} {parsed['Consequence']}")

Parse SnpEff ANN

from cyvcf2 import VCF

def parse_snpeff_ann(ann_string):
    fields = ['Allele', 'Annotation', 'Impact', 'Gene_Name', 'Gene_ID',
              'Feature_Type', 'Feature_ID', 'Transcript_BioType', 'Rank',
              'HGVS_c', 'HGVS_p', 'cDNA_pos', 'CDS_pos', 'Protein_pos', 'Distance']
    values = ann_string.split('|')
    return dict(zip(fields, values[:len(fields)]))

for variant in VCF('snpeff_output.vcf'):
    ann = variant.INFO.get('ANN')
    if ann:
        for transcript in ann.split(','):
            parsed = parse_snpeff_ann(transcript)
            if parsed['Impact'] == 'HIGH':
                print(f"{variant.CHROM}:{variant.POS} {parsed['Gene_Name']} {parsed['Annotation']}")

Complete Annotation Pipeline

Goal: Run a full annotation workflow from normalization through VEP annotation to impact filtering.

Approach: Normalize variants (mandatory -- same variant represented differently gets different annotations), annotate with VEP using MANE Select transcript selection and comprehensive flags, then filter for HIGH/MODERATE impact.

#!/bin/bash
set -euo pipefail

INPUT=$1
REFERENCE=$2
VEP_CACHE=$3
OUTPUT_PREFIX=$4

# Normalize variants (mandatory before annotation)
bcftools norm -f $REFERENCE -m-any $INPUT -Oz -o ${OUTPUT_PREFIX}_norm.vcf.gz
bcftools index ${OUTPUT_PREFIX}_norm.vcf.gz

# VEP annotation with MANE Select transcript selection
# --mane_select: adds MANE Select transcript info
# --pick: selects one consequence per variant (uses MANE Select when available)
# For research (all transcripts): remove --pick, add --per_gene
vep -i ${OUTPUT_PREFIX}_norm.vcf.gz \
    -o ${OUTPUT_PREFIX}_vep.vcf \
    --vcf --cache --offline --dir_cache $VEP_CACHE \
    --assembly GRCh38 --everything --mane_select --pick --fork 4

bgzip ${OUTPUT_PREFIX}_vep.vcf
bcftools index ${OUTPUT_PREFIX}_vep.vcf.gz

# Filter high/moderate impact
bcftools view -i 'INFO/CSQ~"HIGH" || INFO/CSQ~"MODERATE"' \
    ${OUTPUT_PREFIX}_vep.vcf.gz -Oz -o ${OUTPUT_PREFIX}_filtered.vcf.gz

Pathogenicity Predictors

Predictor	Deleterious Threshold	Scope	Notes
SIFT	< 0.05	Missense	Sequence conservation-based; fast but limited
PolyPhen-2 (HDIV)	> 0.957 (probably), > 0.453 (possibly)	Missense	Structure + conservation
CADD	>= 20 (top 1%), >= 30 (top 0.1%)	All variant types	Low specificity (~12%) at default thresholds; good for ranking, poor for binary classification
REVEL	> 0.5 (ClinGen recommended)	Missense	Generally best-performing single missense predictor; ensemble of 13 tools
AlphaMissense	> 0.564 (pathogenic), < 0.340 (benign)	Missense	Protein structure-based (AlphaFold2); MCC=0.81; classified 89% of all possible human missense variants; not trained on pathogenicity labels

No single predictor is sufficient for clinical classification. ACMG/AMP guidelines (PP3/BP4) require that computational evidence be treated as supporting, not standalone. ClinGen recommends REVEL >= 0.644 for PP3 (supporting pathogenic) and REVEL <= 0.290 for BP4 (supporting benign) with strong evidence thresholds at >= 0.773 and <= 0.183 respectively.

Clinical Significance (ClinVar)

Code	Meaning
Pathogenic	Disease-causing
Likely_pathogenic	Probably disease-causing
Uncertain_significance	VUS
Likely_benign	Probably not disease-causing
Benign	Not disease-causing

Quick Reference

Task	Command
Add rsIDs	bcftools annotate -a dbsnp.vcf.gz -c ID in.vcf.gz
VEP annotation	vep -i in.vcf -o out.vcf --vcf --cache --everything
SnpEff annotation	snpEff ann GRCh38.105 in.vcf > out.vcf
Consequences only	bcftools csq -f ref.fa -g genes.gff in.vcf.gz

Related Skills

• variant-calling/variant-normalization - Normalize before annotating (mandatory preprocessing step)
• variant-calling/filtering-best-practices - Filter by annotations and quality metrics
• variant-calling/clinical-interpretation - ACMG classification and clinical reporting
• variant-calling/vcf-basics - Query annotated fields
• variant-calling/vcf-manipulation - Merge and manipulate annotated VCFs
• database-access/entrez-fetch - Download annotation databases (ClinVar, dbSNP)