Use of Next Generation Sequencing in the Diagnosis of Germline Diseases
This guidance provides recommendations for designing, developing, and validating NGS-based tests intended to aid clinicians in diagnosing symptomatic individuals with suspected germline diseases. It specifically excludes NGS-based tests for microbial infection, cell-free DNA testing, direct-to-consumer uses, fetal testing, pre-implantation embryo testing, risk assessment/prediction, RNA sequencing, screening, standalone diagnostics, tumor genome sequencing, and companion/complementary diagnostics.
Recommended Actions
- Define and document specific indications for use and user needs for the test
- Establish and validate test performance characteristics:
- Accuracy (PPA, NPA, TPPV)
- Precision (reproducibility and repeatability)
- Limit of detection
- Analytical specificity
- Implement quality control measures:
- Coverage metrics
- Base quality thresholds
- Test run quality metrics
- Sample acceptance criteria
- Develop comprehensive documentation:
- Test design and validation protocols
- Software components and versions
- Database versions and updates
- Modification procedures
- Test reports format
- Establish procedures for:
- Sample handling and processing
- Test failure management
- Revalidation after modifications
- Database updates
- Create clear labeling and reporting that includes:
- Indications for use
- Test limitations
- Performance characteristics
- Risk mitigation elements
- Clear result reporting format
- Implement a change management system to track and validate modifications
- Develop procedures for continuous monitoring of test performance during clinical use
Key Considerations
Non-clinical testing
- Establish minimum acceptable thresholds for coverage, base quality, and test run quality metrics
- Evaluate precision (reproducibility and repeatability) for both variant and wild-type calls
- Establish and document the minimum/maximum amount of DNA that will enable expected results
- Establish and document analytical specificity using interference, cross-reactivity and cross-contamination metrics
- Validate test performance using well-characterized reference materials or agreed-upon samples
Software
- Document all software components, versions and modifications
- Validate bioinformatics pipeline end-to-end
- Document software versions and traceability
- Specify whether software will run locally or remotely
- Document which databases will be used and their versions
Labelling
- Include indications for use statement with type of sequence variations detected
- List test limitations and risk mitigation elements
- Specify test performance characteristics and metrics
- Provide information about probability of test failure
- Use clear, consistent language throughout reports
- Report variants using widely accepted nomenclature
Safety
- Document specimen handling, preservation, processing, storage and rejection criteria
- Establish quality control acceptance criteria
- Document procedures for test failures
- Validate sample pooling methods if applicable
Other considerations
- Document all modifications to the test
- Prepare detailed SOPs for revalidation after modifications
- Include procedures to account for database updates
- Document performance after any modifications
Relevant Guidances
- Content of Premarket Submissions for Device Software Functions
- Off-The-Shelf Software in Medical Devices: Documentation Requirements for Premarket Submissions
- Software Validation for Medical Device Production, Quality Systems, and Device Components
- Unique Device Identifier (UDI) Form and Content Requirements
- Use of ISO 10993-1 for Biological Evaluation and Testing of Medical Devices
Related references and norms
- CLSI MM09-A2: Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine
- CLSI EP12-A2: User Protocol for Evaluation of Qualitative Test Performance
Original guidance
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