ImmuneRepertoireMrdQcChecker

Immune Repertoire MRD QC

Liquid Biopsy жидкостная биопсия cfDNA ctDNA CTC exosomes NGS qPCR

Utility description: Immune Repertoire MRD QC

Immune Repertoire MRD QC Checker — Immune Repertoire MRD Data Quality Control

ℹ️ Utility performs comprehensive quality verification of TCR/BCR-seq data for MRD monitoring according to EuroClonality-NGS, AIRR Community and NGS-MRD guidelines:
• Sequencing Depth & UMI: Total depth and unique molecule count — foundation of MRD sensitivity.
• PCR Duplication Control: Deduplication rate as library complexity indicator.
• Background Noise: Noise frequency in negative controls — determines detection limit.
• Replicate Reproducibility: Correlation between technical replicates — reliability measure.
• Sensitivity Verification: Comparison of achieved sensitivity with protocol-required level.
• MRD Call Reliability: Minimum read count for reliable MRD positivity call.

⚠️ IMPORTANT:
• MRD-negative result with insufficient sensitivity = FALSE REMISSION.
• MRD-positive call with <10 reads = likely FALSE POSITIVE.
• High background noise (>10⁻⁵) makes reliable MRD determination at 10⁻⁶ impossible.
• Absence of internal sensitivity control prevents assay performance verification.

Usage:
ImmuneRepertoireMrdQcChecker.exe → demo mode (console output)
ImmuneRepertoireMrdQcChecker.exe input.csv output.json → evaluate your data

Input format:
SampleID,PatientID,ReceptorType,Timepoint,TotalReads,MinTotalReads,UniqueMolecules_UMI,MinUniqueMolecules,UMI_Deduplication_Rate,MaxUMI_DedupRate,BackgroundNoise_Frequency,MaxBackgroundNoise,ClonotypeCount_PostDedup,ReplicateCorrelation_R,MinReplicateCorrelation,ControlClonotype_Detected,Sensitivity_Achieved,RequiredSensitivity,MRD_Positive,MRD_CloneFrequency,MRD_CloneReads,MinMRD_CloneReads

Example:
MRD-001,PAT-001,IGH,Post-ind,5200000,1000000,480000,100000,0.65,0.80,2E-6,1E-5,35000,0.96,0.90,true,5E-6,1E-5,false,0,0,10

📍 Scope of Application (Usage Where):
• Hematologic Oncology: MRD monitoring in ALL, CLL, multiple myeloma.
• Clinical Trials: MRD data standardization in multi-center trials.
• Regulatory Submissions: Confirmation of NGS-MRD assay analytical validity.
• Transplant Medicine: Allo-HSCT efficacy assessment by clonotype dynamics.

— WHY IS THIS NEEDED?
MRD status is the strongest prognostic factor in hematology.
False-negative MRD leads to premature therapy cessation and relapse.
False-positive MRD leads to unnecessary toxic consolidation.
Automated QC guarantees that every MRD result is based on sufficient quality data.

⚠️ CRITICAL:
• Total Reads ≥ 1M: Baseline threshold for MRD at 10⁻⁵ level.
• Unique Molecules ≥ 100K: Determines real sensitivity after deduplication.
• Background Noise ≤ 10⁻⁵: Exceedance makes MRD calls unreliable.
• Sensitivity Achieved ≤ Required: MANDATORY for MRD-negative conclusions.
• MRD Clone Reads ≥ 10: Below this threshold, positive call is unreliable.
• Control Clonotype: Absence = unverifiable assay performance.

Key features:
• Eight-parameter MRD QC assessment
• Integration of physical and statistical metrics
• Automatic MRD call reliability verification
• PASS / WARNING / FAIL classification
• Compliance with EuroClonality-NGS and AIRR standards

Critical parameters:
• Total Reads: ≥ 1,000,000
• Unique Molecules: ≥ 100,000
• UMI Dedup Rate: ≤ 80%
• Background Noise: ≤ 1×10⁻⁵
• Replicate Correlation: ≥ 0.90
• Sensitivity Achieved: ≤ Required
• MRD Clone Reads: ≥ 10 (if positive)
• Control Clonotype: Detected

💡 Usage tips:
1. UMI Design: Use UMI length ≥8 nt to minimize collisions at high depth.
2. NTC Controls: Include minimum 3 NTCs per plate for accurate background estimation.
3. Spike-in Controls: Add known clonotypes for sensitivity verification.
4. Replicates: Perform minimum 2 technical replicates for each MRD sample.
5. Thresholds: Adjust MinMRD_CloneReads per panel-specific depth and noise level.

⚠️ Note: This utility assesses TECHNICAL QUALITY of MRD data. Clinical interpretation of MRD status must consider disease type, treatment phase, and prior dynamics. The utility does not replace hematologist expertise.

input.csv

SampleID,PatientID,ReceptorType,Timepoint,TotalReads,MinTotalReads,UniqueMolecules_UMI,MinUniqueMolecules,UMI_Deduplication_Rate,MaxUMI_DedupRate,BackgroundNoise_Frequency,MaxBackgroundNoise,ClonotypeCount_PostDedup,ReplicateCorrelation_R,MinReplicateCorrelation,ControlClonotype_Detected,Sensitivity_Achieved,RequiredSensitivity,MRD_Positive,MRD_CloneFrequency,MRD_CloneReads,MinMRD_CloneReads
MRD-2026-001,ALL-PAT-001,IGH,Post-induction,5200000,1000000,480000,100000,0.65,0.80,2E-6,1E-5,35000,0.96,0.90,true,5E-6,1E-5,false,0,0,10
MRD-2026-002,ALL-PAT-002,TRB,Follow-up,350000,1000000,42000,100000,0.88,0.80,5E-5,1E-5,8500,0.72,0.90,false,2E-4,1E-5,true,3E-5,5,10
MRD-2026-003,MM-PAT-003,IGK,Post-ASCT,2800000,1000000,250000,100000,0.70,0.80,8E-6,1E-5,28000,0.93,0.90,false,8E-6,1E-5,false,0,0,10

URS & FS — User Requirements and Functional Specification

This document describes the controlled interface and behaviour of ImmuneRepertoireMrdQcChecker for Immune Repertoire MRD QC Checker.

Domain limits URS input.csv FS IQ/OQ/PQ

Domain limits and critical parameters

Key fragments from the source description are shown below. Before production use, limits must be verified against the approved specification, registration dossier and local SOPs.

• Background Noise: Noise frequency in negative controls — determines detection limit.
• Sensitivity Verification: Comparison of achieved sensitivity with protocol-required level.
• MRD-positive call with <10 reads = likely FALSE POSITIVE.
• High background noise (>10⁻⁵) makes reliable MRD determination at 10⁻⁶ impossible.
⚠️ CRITICAL:
• Total Reads ≥ 1M: Baseline threshold for MRD at 10⁻⁵ level.
• Unique Molecules ≥ 100K: Determines real sensitivity after deduplication.
• Background Noise ≤ 10⁻⁵: Exceedance makes MRD calls unreliable.
• Sensitivity Achieved ≤ Required: MANDATORY for MRD-negative conclusions.
• MRD Clone Reads ≥ 10: Below this threshold, positive call is unreliable.
Critical parameters:
• Total Reads: ≥ 1,000,000
• Unique Molecules: ≥ 100,000
• UMI Dedup Rate: ≤ 80%
• Background Noise: ≤ 1×10⁻⁵
• Replicate Correlation: ≥ 0.90
• Sensitivity Achieved: ≤ Required
• MRD Clone Reads: ≥ 10 (if positive)

URS — User Requirements Specification

ID	Requirement	Criticality	Acceptance criterion
URS-001	The utility shall accept an input.csv file for Immune Repertoire MRD QC Checker with headers defined in the data contract.	High	The file is processed without manual header editing.
URS-002	The utility shall perform deterministic QC evaluation without machine learning and without probabilistic conformance decisions.	High	Identical input data, rule version and configuration produce reproducible results.
URS-003	The utility shall validate mandatory fields, data types, ranges, units and domain plausibility.	High	Schema, conversion and range errors are explicitly reported.
URS-004	The utility shall apply domain limits and rules from the description, approved specification, registration dossier and local SOPs.	High	Each check has PASS/WARNING/FAIL and a clear message.
URS-005	The utility shall generate output.json with machine-readable results, source values, warnings, failures and critical findings.	High	JSON is suitable for LIMS/ELN/MES integration and QA/QC review.
URS-006	The utility shall preserve traceability between batch/sample, input file, applied rules and final status.	High	Output contains identifiers, checked parameters and audit metadata.
URS-007	The documentation shall support IQ/OQ/PQ, CSV/CSA and review by internal QA or inspectors.	Medium	URS, FS, input/output contract and test scenarios are supplied with the utility.
URS-008	The utility shall be used as a QC decision-support tool and not as a substitute for approved specifications and QA/QP release decision.	Medium	Documentation states change control and limit-verification expectations.

input.csv contract

#	Field	Type	Sample	Purpose
1	`SampleID`	string / controlled vocabulary	`MRD-2026-001`	Sample or laboratory specimen identifier.
2	`PatientID`	string / controlled vocabulary	`ALL-PAT-001`	Controlled input parameter for deterministic QC rules.
3	`ReceptorType`	string / controlled vocabulary	`IGH`	Controlled input parameter for deterministic QC rules.
4	`Timepoint`	string / controlled vocabulary	`Post-induction`	Time parameter for process, incubation, storage or analytical workflow.
5	`TotalReads`	decimal	`5200000`	Controlled input parameter for deterministic QC rules.
6	`MinTotalReads`	decimal	`1000000`	Controlled input parameter for deterministic QC rules.
7	`UniqueMolecules_UMI`	decimal	`480000`	Controlled input parameter for deterministic QC rules.
8	`MinUniqueMolecules`	decimal	`100000`	Controlled input parameter for deterministic QC rules.
9	`UMI_Deduplication_Rate`	decimal	`0.65`	Controlled input parameter for deterministic QC rules.
10	`MaxUMI_DedupRate`	decimal	`0.80`	Controlled input parameter for deterministic QC rules.
11	`BackgroundNoise_Frequency`	decimal	`2E-6`	Controlled input parameter for deterministic QC rules.
12	`MaxBackgroundNoise`	decimal	`1E-5`	Controlled input parameter for deterministic QC rules.
13	`ClonotypeCount_PostDedup`	integer / decimal	`35000`	Count parameter used for microbiological, particulate or cellular control.
14	`ReplicateCorrelation_R`	decimal	`0.96`	Controlled input parameter for deterministic QC rules.
15	`MinReplicateCorrelation`	integer / decimal	`0.90`	Controlled input parameter for deterministic QC rules.
16	`ControlClonotype_Detected`	string / controlled vocabulary	`true`	Control point/control sample for assay run validity.
17	`Sensitivity_Achieved`	decimal	`5E-6`	Controlled input parameter for deterministic QC rules.
18	`RequiredSensitivity`	decimal	`1E-5`	Controlled input parameter for deterministic QC rules.
19	`MRD_Positive`	string / controlled vocabulary	`false`	Controlled input parameter for deterministic QC rules.
20	`MRD_CloneFrequency`	decimal	`0`	Controlled input parameter for deterministic QC rules.
21	`MRD_CloneReads`	decimal	`0`	Controlled input parameter for deterministic QC rules.
22	`MinMRD_CloneReads`	decimal	`10`	Controlled input parameter for deterministic QC rules.

SampleID,PatientID,ReceptorType,Timepoint,TotalReads,MinTotalReads,UniqueMolecules_UMI,MinUniqueMolecules,UMI_Deduplication_Rate,MaxUMI_DedupRate,BackgroundNoise_Frequency,MaxBackgroundNoise,ClonotypeCount_PostDedup,ReplicateCorrelation_R,MinReplicateCorrelation,ControlClonotype_Detected,Sensitivity_Achieved,RequiredSensitivity,MRD_Positive,MRD_CloneFrequency,MRD_CloneReads,MinMRD_CloneReads
MRD-2026-001,ALL-PAT-001,IGH,Post-induction,5200000,1000000,480000,100000,0.65,0.80,2E-6,1E-5,35000,0.96,0.90,true,5E-6,1E-5,false,0,0,10
MRD-2026-002,ALL-PAT-002,TRB,Follow-up,350000,1000000,42000,100000,0.88,0.80,5E-5,1E-5,8500,0.72,0.90,false,2E-4,1E-5,true,3E-5,5,10
MRD-2026-003,MM-PAT-003,IGK,Post-ASCT,2800000,1000000,250000,100000,0.70,0.80,8E-6,1E-5,28000,0.93,0.90,false,8E-6,1E-5,false,0,0,10

Input validation rules

ID	Field	Rule	Criticality
VR-001	`SampleID`	The field shall match an approved dictionary or accepted string representation.	High
VR-002	`PatientID`	The field shall match an approved dictionary or accepted string representation.	High
VR-003	`ReceptorType`	The field shall match an approved dictionary or accepted string representation.	High
VR-004	`Timepoint`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-005	`TotalReads`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-006	`MinTotalReads`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-007	`UniqueMolecules_UMI`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-008	`MinUniqueMolecules`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-009	`UMI_Deduplication_Rate`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-010	`MaxUMI_DedupRate`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-011	`BackgroundNoise_Frequency`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-012	`MaxBackgroundNoise`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-013	`ClonotypeCount_PostDedup`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-014	`ReplicateCorrelation_R`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-015	`MinReplicateCorrelation`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-016	`ControlClonotype_Detected`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-017	`Sensitivity_Achieved`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-018	`RequiredSensitivity`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-019	`MRD_Positive`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-020	`MRD_CloneFrequency`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-021	`MRD_CloneReads`	The field shall match an approved dictionary or accepted string representation.	Medium
VR-022	`MinMRD_CloneReads`	The field shall match an approved dictionary or accepted string representation.	Medium

FS — Functional Specification

ID	Function	Implementation
FS-001	CLI execution	Support execution modes: demo mode without arguments and production mode input.csv output.json.
FS-002	CSV import	Read input.csv in UTF-8/CSV-compatible format and validate header and expected columns.
FS-003	Schema validation	Check mandatory fields, column count, unknown key fields and empty mandatory values.
FS-004	Type conversion	Convert numeric, flag and text values; invalid format is recorded as a row-level error.
FS-005	Domain rule engine	Apply rules for Immune Repertoire MRD QC Checker, including critical limits from the description and approved specification.
FS-006	Status aggregation	Produce final status: FAIL for critical failure, WARNING for non-critical deviation, PASS for conformance.
FS-007	JSON export	Write output.json with detailed checks, source values, warnings, failures and critical findings.
FS-008	Audit support	Keep result structure suitable for review, deviation investigation and calculation reproduction.
FS-009	Integration contract	Support the scenario LIMS/ELN/MES → input.csv → utility → output.json → portal/admin review.
FS-010	Error handling	Return explicit messages for missing file, empty CSV, invalid schema, output write failure and invalid format.

Example output.json

{
  "utilityId": "immunerepertoiremrdqcchecker",
  "utilityFolder": "ImmuneRepertoireMrdQcChecker",
  "package": "LiquidBiopsy",
  "overallStatus": "PASS|WARNING|FAIL",
  "sourceFile": "input.csv",
  "processedAtUtc": "2026-06-10T00:00:00Z",
  "checks": [
    {
      "parameter": "SampleID",
      "value": "MRD-2026-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-001"
    },
    {
      "parameter": "PatientID",
      "value": "ALL-PAT-001",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-002"
    },
    {
      "parameter": "ReceptorType",
      "value": "IGH",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-003"
    },
    {
      "parameter": "Timepoint",
      "value": "Post-induction",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-004"
    },
    {
      "parameter": "TotalReads",
      "value": "5200000",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-005"
    },
    {
      "parameter": "MinTotalReads",
      "value": "1000000",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-006"
    },
    {
      "parameter": "UniqueMolecules_UMI",
      "value": "480000",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-007"
    },
    {
      "parameter": "MinUniqueMolecules",
      "value": "100000",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-008"
    },
    {
      "parameter": "UMI_Deduplication_Rate",
      "value": "0.65",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-009"
    },
    {
      "parameter": "MaxUMI_DedupRate",
      "value": "0.80",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-010"
    },
    {
      "parameter": "BackgroundNoise_Frequency",
      "value": "2E-6",
      "status": "PASS|WARNING|FAIL",
      "message": "Deterministic rule-based check result",
      "ruleReference": "FS-RULE-011"
    },
    {
      "parameter": "MaxBackgroundNoise",
      "value": "1E-5",
      "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": "ImmuneRepertoireMrdQcChecker.documentation.html"
  }
}

Traceability matrix

URS	FS	Test	Evidence
URS-001	FS-001, FS-002	OQ-001	Verify execution and import of valid input.csv.
URS-002	FS-005, FS-006	OQ-004	Repeat the same dataset and compare output.json.
URS-003	FS-003, FS-004, FS-010	OQ-002, OQ-003	Verify missing columns and invalid types.
URS-004	FS-005, FS-006	OQ-004, PQ-001	Verify critical deviations on real/boundary data.
URS-005	FS-007, FS-009	OQ-005	Verify JSON schema and downstream-system suitability.
URS-006	FS-008	OQ-006	Verify identifiers and audit metadata.
URS-007	FS-008, FS-010	IQ-001, OQ-007	Verify documentation completeness and control evidence.
URS-008	FS-005, FS-008	PQ-002	Verify review workflow and no replacement of QA decision.

IQ/OQ/PQ test scenarios

ID	Scenario	Expected result
IQ-001	Verify executable, input.csv, documentation and checksum availability.	Delivery set is complete; version is recorded.
OQ-001	Valid sample row from input.csv.	PASS or acceptable WARNING according to rules.
OQ-002	Remove a mandatory CSV column.	Schema error or FAIL with missing-column reference.
OQ-003	Place a non-numeric value into a numeric field.	Type-conversion error with row/field reference.
OQ-004	Set a critical parameter outside the limit.	FAIL and critical finding.
OQ-005	Verify output.json structure.	All mandatory sections are present and JSON is valid.
OQ-006	Verify batch/sample traceability.	Input and result identifiers match.
PQ-001	Verify 3–5 real user batches/samples.	Result is confirmed by QC/QA review.
PQ-002	Verify deviation workflow and manual QA decision.	Utility supports review but does not replace approved decision.

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.

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