Personal-Genome-Pipeline

Step 31: Variant Prioritization (slivar)

Prioritizes clinically interesting variants using tiered filters and detects compound heterozygote candidates. Optionally annotates results with gnomAD gene constraint metrics.

Why

A typical VEP-annotated VCF contains thousands of MODERATE/HIGH impact variants. Most are common population variants or benign polymorphisms. This step filters down to the variants most likely to be clinically relevant using a three-tier approach, then flags potential compound heterozygotes that could cause autosomal recessive disease.

slivar (by Brent Pedersen, author of vcfanno, mosdepth, duphold) is a streaming VCF filter that replaces the unmaintained GEMINI database approach. It uses JS expressions for flexible filtering without loading variants into a database.

Prerequisites

• VEP-annotated VCF from step 13, or vcfanno-enriched VCF from step 30 (recommended)
• Optional: gnomAD v4.1 gene constraint TSV at ${GENOME_DIR}/annotations/gnomad_v4.1_constraint.tsv

Docker Images

quay.io/biocontainers/slivar:0.3.3--h5f107b1_0    # compound het detection
staphb/bcftools:1.21                                # variant filtering via split-vep

Usage

export GENOME_DIR=/path/to/your/data
./scripts/31-slivar.sh your_sample

Filter Tiers

Tier 1: rare_high

• PASS variants with HIGH VEP impact (stop-gain, frameshift, splice donor/acceptor)
• gnomAD allele frequency < 1% (or missing)

Tier 2: rare_moderate_deleterious

• PASS variants with MODERATE VEP impact (missense, in-frame indel)
• gnomAD allele frequency < 1% (or missing)
• At least one deleterious predictor hit (if vcfanno annotations available):
  • CADD PHRED >= 20 (SNV and/or indel tags, whichever are present)
  • REVEL >= 0.5
  • AlphaMissense “likely_pathogenic”
  • SpliceAI annotation present (presence check only — bcftools cannot parse the pipe-delimited delta scores; threshold filtering at >= 0.2 is done in step 23)
• Without vcfanno: all rare MODERATE variants included (same as step 23)
• Only annotation tags that exist in the VCF header are referenced — partial installs (e.g., CADD SNVs only) work correctly

Tier 3: clinvar_pathogenic

• PASS variants with ClinVar pathogenic or likely_pathogenic (from VEP CLIN_SIG field)
• Excludes conflicting_interpretations_of_pathogenicity (the substring match ~"pathogenic" would otherwise include these)
• No frequency filter (pathogenic variants can be common carriers)

All tiers are merged and deduplicated into a single prioritized VCF.

Compound Heterozygote Detection

slivar’s compound-hets command groups heterozygous variants by gene from the prioritized VCF and reports pairs that could form compound heterozygotes (two different damaging variants in the same gene, one from each parent). It requires a PED file (--ped) describing sample relationships. For singleton samples (no trio), the --allow-non-trios flag is required.

The command outputs VCF to stdout with INFO/slivar_comphet annotations linking partner variants. Each VCF record represents a unique variant; the slivar_comphet field lists all its compound-het partners (format: sample/GENE/PAIR_ID/chrom/pos/ref/alt, comma-separated). A gene with N variants produces up to C(N,2) pairs but only N VCF records. The script counts unique pair IDs from this field and exports a human-readable TSV with columns: GENE, CHROM, POS, REF, ALT, IMPACT, Consequence, GT – sorted by gene so that compound-het partners appear in consecutive rows.

Important: With single-sample unphased data, these are candidates only. The two variants might be on the same haplotype (cis) rather than different haplotypes (trans). Trio data or read-backed phasing is needed to confirm true compound hets.

Gene Constraint Enrichment

If gnomad_v4.1_constraint.tsv is available, the summary TSV is enriched with per-gene constraint metrics:

Column	Description	Threshold
LOEUF	Loss-of-function observed/expected upper bound	< 0.35 = constrained
pLI	Probability of LoF intolerance	> 0.9 = constrained
mis_z	Missense Z-score	> 3.09 = constrained
CONSTRAINED	YES if LOEUF < 0.35 or pLI > 0.9	Flag column

Variants in constrained genes are more likely to be pathogenic – these genes are under strong purifying selection against damaging variants.

Output

File	Description
slivar/${SAMPLE}_prioritized.vcf.gz	All prioritized variants (merged, deduplicated)
slivar/${SAMPLE}_slivar_summary.tsv	Human-readable table with gene constraint
slivar/${SAMPLE}_compound_hets.vcf.gz	Compound het candidate variants (VCF)
slivar/${SAMPLE}_compound_hets.tsv	Compound het candidates (human-readable TSV)
slivar/${SAMPLE}_rare_high.vcf.gz	Tier 1: HIGH impact
slivar/${SAMPLE}_rare_moderate_del.vcf.gz	Tier 2: MODERATE + deleterious
slivar/${SAMPLE}_clinvar_path.vcf.gz	Tier 3: ClinVar P/LP

Runtime

~5-10 minutes. Most time is spent on bcftools split-vep filtering.

Interpretation

A typical 30X WGS sample produces:

• rare_high: 50-200 variants (loss-of-function in rare alleles)
• rare_moderate_deleterious: 200-1,000 variants (depends on predictor availability)
• clinvar_pathogenic: 0-10 variants (most are heterozygous carriers)
• compound het candidates: 1,000-2,000 pairs across 100-200 genes (combinatorial: N variants in a gene = N*(N-1)/2 pairs; most are false positives in unphased data)

Focus review on:

1. Variants in constrained genes (CONSTRAINED=YES)
2. Homozygous rare HIGH/MODERATE variants (potential recessive disease)
3. Compound het pairs in known disease genes
4. Any ClinVar pathogenic variants, especially in dominant disease genes

See docs/interpreting-results.md for pathogenicity score thresholds.

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