QiaSeqLibraryQcChecker

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of QiaSeqLibraryQcChecker for QIAseq Library QC Checker.

Domain limits and critical parameters

• Performs comprehensive quality verification of QIAseq libraries per Qiagen QIAseq User Manual, CAP/CLIA, FDA NGS guidance and SEQC2 standards: library physical parameters (concentration ≥2nM, size peak ±50bp, adapter dimer ≤5%, primer dimer ≤10% for amplicon), UMI metrics (incorporation rate ≥80%, unique UMIs ≥10K, diversity index ≥0.01, mean family size ≥3.0, collision rate ≤5%, post-dedup duplication ≤20%), enrichment metrics (on-target ≥60%, uniformity ≥75%, depth ≥500×, fold-80 ≤2.5×, zero c
• ⚠️ Critical: UMI incorporation <80% = ERROR CORRECTION UNRELIABLE; QIAseq advantage lost. High pre-dedup duplication is NORMAL for QIAseq; POST-DEDUP metric matters. Mean family size <3 = consensus built from <3 molecules; errors not corrected. Adapter dimer >5% competes during clustering; repeat SPRI required. QIAseq spike-in failure indicates ligation or RT step issue. For amplicon panels, primer dimer and amplicon balance are specific critical parameters. Thresholds are panel-dependent; use Q

URS — User Requirements Specification

input.csv contract

SampleID,PanelType,PanelName,KitVersion,SequencingPlatform,LibraryConcentration_nM,MinLibraryConcentration_nM,LibrarySize_Peak_bp,ExpectedSize_Peak_bp,SizeTolerance_bp,AdapterDimer_Percent,MaxAdapterDimer_Percent,PrimerDimer_Percent,MaxPrimerDimer_Percent,UMI_IncorporationRate_Percent,MinUMI_IncorporationRate_Percent,UniqueUMIs_Count,MinUniqueUMIs,UMIDiversity_Index,MinUMIDiversity_Index,PreDedup_DuplicationRate_Percent,PostDedup_DuplicationRate_Percent,MeanFamilySize,MinMeanFamilySize,UMICollisionRate_Percent,MaxUMICollisionRate_Percent,OnTargetRate_Percent,MinOnTargetRate_Percent,Uniformity_Percent,MinUniformity_Percent,MeanTargetDepth_X,MinMeanTargetDepth_X,Fold80_BasePenalty,MaxFold80,CriticalRegions_BelowThreshold,TotalCriticalRegions,AmpliconBalance_CV_Percent,MaxAmpliconBalance_CV_Percent,AmpliconsBelowMinCoverage,TotalAmplicons,PositiveControl_Pass,NegativeControl_Pass,NoTemplateControl_Pass,SpikeIn_Control_Pass,Molarity_nM_Final,MinMolarity_ForSequencing_nM,IndexSequence_Verified,PhiX_SpikeIn_Planned
QS-TDNA-2026-001,Targeted_DNA,QIAseq_HPV_Panel,v2.1,MiSeq,15.2,2.0,345,350,50,1.2,5.0,0,10.0,94.5,80,85000,10000,0.085,0.01,45.0,2.1,4.8,3.0,1.2,5.0,88.0,60,91.0,75,2500,500,1.4,2.5,0,45,0,30,0,0,true,true,true,true,12.5,2.0,true,true
QS-TDNA-2026-002,Targeted_DNA,QIAseq_BRCA_Panel,v2.1,NextSeq,3.8,2.0,180,350,50,12.5,5.0,0,10.0,62.0,80,5200,10000,0.003,0.01,78.0,35.0,1.8,3.0,8.5,5.0,52.0,60,65.0,75,180,500,3.8,2.5,8,32,0,30,0,0,true,false,true,false,2.8,2.0,true,true

Input validation rules

FS — Functional Specification

Example output.json

{
  "utilityId": "qiaseqlibraryqcchecker",
  "utilityFolder": "QiaSeqLibraryQcChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "QS-TDNA-2026-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "PanelType",
      "value": "Targeted_DNA",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-002"
    },
    {
      "parameter": "PanelName",
      "value": "QIAseq_HPV_Panel",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-003"
    },
    {
      "parameter": "KitVersion",
      "value": "v2.1",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "SequencingPlatform",
      "value": "MiSeq",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "LibraryConcentration_nM",
      "value": "15.2",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "MinLibraryConcentration_nM",
      "value": "2.0",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "LibrarySize_Peak_bp",
      "value": "345",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "ExpectedSize_Peak_bp",
      "value": "350",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "SizeTolerance_bp",
      "value": "50",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "AdapterDimer_Percent",
      "value": "1.2",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "MaxAdapterDimer_Percent",
      "value": "5.0",
      "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": "QiaSeqLibraryQcChecker.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.