MethylationPanelRunQcChecker

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of MethylationPanelRunQcChecker for Methylation Panel Run QC Checker.

Domain limits and critical parameters

• Performs quality verification of methylation panel sequencing runs with bisulfite-specific metrics per ENCODE, CAP/CLIA and IHEC standards: bisulfite conversion rate (≥99%), non-CpG C retention (≤1%), adapted sequencing thresholds for BS-seq (Q30 ≥70%, on-target ≥50%, uniformity ≥70%, duplication ≤60%), CpG coverage completeness (≥85%), dual methylation controls (fully methylated ≥95%, unmethylated ≤5%), fragment profile validation, and library complexity assessment. Three-tier classification: P
• ⚠️ Critical: Incomplete bisulfite conversion mimics FALSE HYPERMETHYLATION genome-wide. Non-CpG retention is more reliable indicator than overall conversion. BS-seq thresholds DIFFER from standard NGS — do not apply standard filters. Methylation controls are MANDATORY in every run.

URS — User Requirements Specification

input.csv contract

RunID,PanelName,Platform,ConversionMethod,BisulfiteConversion_Rate,MinConversion_Rate,NonCpG_CytosineRetention_Percent,MaxNonCpG_Retention_Percent,PercentQ30,MinQ30,MeanTargetDepth_X,MinMeanTargetDepth_X,OnTargetRate_Percent,MinOnTargetRate_Percent,Uniformity_Percent,MinUniformity_Percent,DuplicationRate_Percent,MaxDuplicationRate_Percent,CpG_Sites_Covered,CpG_Sites_Target_Total,CpG_Coverage_Completeness_Percent,MinCpG_Completeness_Percent,FragmentSize_Peak_bp,FragmentProfile_Valid,FullyMethylated_Control_Pass,FullyMethylated_Control_Level,MinFullyMethylated_Level,Unmethylated_Control_Pass,Unmethylated_Control_Level,MaxUnmethylated_Level,NTC_Pass,UniqueMolecules_Count,MinUniqueMolecules,LibraryComplexity_Score,MinLibraryComplexity_Score
METHYL-RUN-2026-001,EpiMark_500,NovaSeq 6000,Bisulfite,0.997,0.99,0.3,1.0,82.0,70,250,100,72.0,50,85.0,70,35.0,60,48500,50000,97.0,85,165,true,true,0.98,0.95,true,0.02,0.05,true,85000,10000,0.78,0.5
METHYL-RUN-2026-002,EpiMark_500,NovaSeq 6000,Bisulfite,0.965,0.99,3.5,1.0,68.0,70,80,100,45.0,50,58.0,70,72.0,60,32000,50000,64.0,85,140,false,false,0.82,0.95,false,0.12,0.05,true,5200,10000,0.32,0.5

Input validation rules

FS — Functional Specification

Example output.json

{
  "utilityId": "methylationpanelrunqcchecker",
  "utilityFolder": "MethylationPanelRunQcChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "RunID",
      "value": "METHYL-RUN-2026-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "PanelName",
      "value": "EpiMark_500",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-002"
    },
    {
      "parameter": "Platform",
      "value": "NovaSeq 6000",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-003"
    },
    {
      "parameter": "ConversionMethod",
      "value": "Bisulfite",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "BisulfiteConversion_Rate",
      "value": "0.997",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "MinConversion_Rate",
      "value": "0.99",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "NonCpG_CytosineRetention_Percent",
      "value": "0.3",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "MaxNonCpG_Retention_Percent",
      "value": "1.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "PercentQ30",
      "value": "82.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "MinQ30",
      "value": "70",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "MeanTargetDepth_X",
      "value": "250",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "MinMeanTargetDepth_X",
      "value": "100",
      "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": "MethylationPanelRunQcChecker.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.