Next Generation Sequencing (NGS) Worksheets

Next Generation Sequencing (NGS) Worksheets

• Design, development, and routine operation of clinical NGS tests remains challenging as regulatory requirements are vague, and most guidance documents focus on describing requirements without providing detailed instructions on how to translate them into a viable clinical test. In 2018, the College of American Pathologists (CAP) with representation from the Association for Molecular Pathologists (AMP) recognized the need to modernize guidance and created a set of structured worksheets that that guide the user through the entire life cycle of an NGS test (with a focus on germline applications) (https://doi.org/10.1016/j.jmoldx.2018.11.004 ). The initial set has since been refined and expanded to seven worksheets through a partnership with the Clinical and Laboratory Standards Institute (CLSI), which incorporated these worksheets into their MM09 guideline.
• Clinical NGS continues to evolve quickly and because of the diversity of methods, worksheets were intentionally designed to provide concepts and teaching examples reflecting commonly used NGS applications rather than prescriptive performance standards.

CLSI Guideline:

• Title: MM09, Human Genetic and Genomic Testing Using Traditional and High-Throughput Nucleic Acid Sequencing Methods
• Tagline: This guideline in conjunction with instructional worksheets and educational examples provides step-by-step recommendations for designing, testing, validating, reporting, and continual quality management of clinical tests based on next-generation sequencing and Sanger sequencing.
• Suggested Citation: Clinical and Laboratory Standards Institute (CLSI). Human Genetic and Genomic Testing Using Traditional and High-Throughput Nucleic Acid Sequencing Methods. 3rd ed. CLSI guideline MM09. Clinical and Laboratory Standards Institute, USA, 2023.
• Link to CLSI MM09 Standard: https://clsi.org/standards/products/molecular-diagnostics/documents/mm09/

Outlook:

• The series of worksheets available here will undergo regular review and updating and will be expanded to cover additional NGS applications over time.
• Content in progress: Bioinformatics: Principles and Practice for Human Genetics and Oncology (CLSI), Somatic Applications of NGS (CAP)

The seven worksheets are:

Test Familiarization

Focuses on strategic considerations prior to initiating test development in a clinical laboratory.

Download the Test Familiarization NGS Worksheet

Test Content Design

Guides through assembling critical information for genes, disorders, and key variants to ensure clinical validity. Includes considerations on identifying problem regions, correlating those with clinical requirements, and provides a decision matrix for ensuring coverage of all critical regions. Also contains an approach and resources for selecting adequate reference materials for analytical validation.

Download the Test Content Design NGS Worksheet

Assay Design and Optimization

Translates test design requirements into an initial assay design, including defining the coverage over the target regions, capture and sequencing methodologies, as well as any supplementary assays, as needed.

Download the Assay Design and Optimization NGS Worksheet

Test Validation

Describes analytical performance metrics in addition to associated formulas, suggested reference materials, and calculators. Also included are example worksheets on validation study design and subsequent data analysis.

Download the Test Validation NGS Worksheet

Quality Management

Provides an overview of the procedure monitors for the pre-analytical, analytical, and post-analytical phases of NGS-based testing.

Download the Quality Management NGS Worksheet

Bioinformatics and IT

Provides an overview on the critical considerations for the computational infrastructure as well as selection and validation of informatics approaches for tertiary processing of samples (NOTE: the initial aspects of bioinformatics are covered in previous sections).

Download the Bioinformatics and IT NGS Worksheet

Interpretation and Reporting

Contains considerations and requirements for interpretation and reporting of variants identified using germline NGS testing (including filtration approaches, tools and strategies for challenging scenarios, considerations for variant reclassification, reanalysis strategies for exome/genome testing, and a list of databases and software tools)

Download the Interpretation and Reporting NGS Worksheet