VariantAlleleFractionGateChecker

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of VariantAlleleFractionGateChecker for Variant Allele Fraction Gate Checker.

Domain limits and critical parameters

• • LOD/LOQ Comparison: Comparison of observed VAF with validated limits of detection and quantification.
• • Purity-Adjusted Expectation: Correction of expected minimum VAF for tumor purity and ploidy.
• • Germline Suspicion Flag: Identification of variants with VAF ~50%/~100% in somatic samples.
• • At 20% tumor purity, heterozygous somatic mutation has expected VAF ~10%, not 50%.
• • Fixed VAF=5% threshold will discard true variants in low-purity samples.
• • Binomial p-value > 0.05 means alt reads may be explained by sequencing error.
• SampleID,VariantID,Gene,VariantType,Context,ObservedVAF_Percent,TotalDepth,AltReads,TumorPurity_Percent,Ploidy,AssayLOD_Percent,AssayLOQ_Percent,ExpectedMinVAF_PurityAdjusted,BinomialPValue,MaxBinomialPValue,IsKnownHotspot,HasStrandBias,IsInHomopolymer,GnomAD_AF,IsGermlineByVAF
• TUMOR-001,KRAS:c.35G>A,KRAS,SNV,Somatic_Tissue,8.5,450,38,20,2.0,5.0,10.0,7.5,1.2E-12,0.05,true,false,false,0.00001,false
• • Clinical Oncogenomics: Filtering somatic variants in FFPE samples with variable purity.
• • Liquid Biopsy: Validation of ultra-rare variants (VAF <1%) in cfDNA.
• • MRD Monitoring: Distinguishing true signal from noise at sensitivity limit.
• Naive fixed-threshold filtering loses true variants in low-purity samples and passes artifacts in high-depth data.
• This is critically important for liquid biopsy sensitivity and tissue testing specificity.
• ⚠️ CRITICAL:
• • Purity Adjustment: MANDATORY for tissue somatic samples. Without it = systematic variant loss.
• • Binomial P ≤ 0.05: Without statistical significance, VAF is meaningless regardless of absolute value.
• • Alt Reads ≥ 3: Absolute minimum for any call. 1-2 reads = artifact.
• • Germline Suspicion: VAF ~50% in somatic sample requires paired normal verification.

URS — User Requirements Specification

input.csv contract

SampleID,VariantID,Gene,VariantType,Context,ObservedVAF_Percent,TotalDepth,AltReads,TumorPurity_Percent,Ploidy,AssayLOD_Percent,AssayLOQ_Percent,ExpectedMinVAF_PurityAdjusted,BinomialPValue,MaxBinomialPValue,IsKnownHotspot,HasStrandBias,IsInHomopolymer,GnomAD_AF,IsGermlineByVAF
TUMOR-001,KRAS:c.35G>A,KRAS,SNV,Somatic_Tissue,8.5,450,38,20,2.0,5.0,10.0,7.5,1.2E-12,0.05,true,false,false,0.00001,false
LB-001,EGFR:c.2573T>G,EGFR,SNV,Somatic_LiquidBiopsy,0.25,25000,62,100,2.0,0.5,1.0,0.5,3.5E-8,0.05,true,false,false,0.0,false
TUMOR-002,PTEN:c.388C>T,PTEN,Indel,Somatic_Tissue,6.2,180,11,45,2.0,5.0,10.0,18.0,0.08,0.05,false,true,true,0.0001,false

Input validation rules

FS — Functional Specification

Example output.json

{
  "utilityId": "variantallelefractiongatechecker",
  "utilityFolder": "VariantAlleleFractionGateChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "TUMOR-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "VariantID",
      "value": "KRAS:c.35G>A",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-002"
    },
    {
      "parameter": "Gene",
      "value": "KRAS",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-003"
    },
    {
      "parameter": "VariantType",
      "value": "SNV",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "Context",
      "value": "Somatic_Tissue",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "ObservedVAF_Percent",
      "value": "8.5",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "TotalDepth",
      "value": "450",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "AltReads",
      "value": "38",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "TumorPurity_Percent",
      "value": "20",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "Ploidy",
      "value": "2.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "AssayLOD_Percent",
      "value": "5.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "AssayLOQ_Percent",
      "value": "10.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-012"
    }
  ],
  "criticalFindings": [],
  "warnings": [],
  "audit": {
    "inputHash": "sha256:<calculated at runtime>",
    "rulesVersion": "<utility executable version>",
    "documentation": "VariantAlleleFractionGateChecker.documentation.html"
  }
}

Traceability matrix

IQ/OQ/PQ test scenarios

QA/QC and change control

• Do not rename columns without updating validator, documentation and test set.
• Retain input.csv, output.json, executable version and checksum.
• Before production use, perform IQ/OQ/PQ or equivalent CSV/CSA verification.
• Critical limits shall be verified against the approved specification, registration dossier and local SOPs.
• The utility provides structured QC decision support; final release decision remains with QA/QP and approved procedures.