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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.

  1. Define and document specific indications for use and user needs for the test
  2. Establish and validate test performance characteristics:
    • Accuracy (PPA, NPA, TPPV)
    • Precision (reproducibility and repeatability)
    • Limit of detection
    • Analytical specificity
  3. Implement quality control measures:
    • Coverage metrics
    • Base quality thresholds
    • Test run quality metrics
    • Sample acceptance criteria
  4. Develop comprehensive documentation:
    • Test design and validation protocols
    • Software components and versions
    • Database versions and updates
    • Modification procedures
    • Test reports format
  5. Establish procedures for:
    • Sample handling and processing
    • Test failure management
    • Revalidation after modifications
    • Database updates
  6. Create clear labeling and reporting that includes:
    • Indications for use
    • Test limitations
    • Performance characteristics
    • Risk mitigation elements
    • Clear result reporting format
  7. Implement a change management system to track and validate modifications
  8. 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

  • CLSI MM09-A2: Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine
  • CLSI EP12-A2: User Protocol for Evaluation of Qualitative Test Performance

Original guidance

  • Use of Next Generation Sequencing in the Diagnosis of Germline Diseases
  • HTML / PDF
  • Issue date: 2018-04-13
  • Last changed date: 2020-03-02
  • Status: FINAL
  • Official FDA topics: Medical Devices, Biologics
  • ReguVirta summary file ID: 6ddedb8d4e342414e4fcf99f94f6eb56
This post is licensed under CC BY 4.0 by the author.