The Centers for Disease Control and Prevention and the Association of Public Health Laboratories’ Next Generation Sequencing (NGS) Quality Initiative (QI) developed “A Pathway to Quality-Focused Testing” (Pathway). The Pathway is intended to support laboratory personnel responsible for validation, continued testing, and maintenance of NGS workflows. It provides an interactive method to monitor progress and track task completion during a method validation and could be presented to laboratory leadership (or designee) when reviewing validation plans or monitoring progress. The Pathway is intended as one option for approaching method validation. Use does not ensure a successful validation. This Pathway is not intended as a stand-alone document for developing an NGS Quality Management System (QMS) but as a supplement to other published tools and resources that address these topics in more depth. This document identifies additional NGS-related tools and resources that provide additional information and examples (e.g., precision, accuracy). Please visit https://www.cdc.gov/lab-quality/php/ngs-quality-intiative/qms-tools-resources.html. It is expected that each laboratory will use this Pathway, in whole or in individual phases, to guide NGS method validation according to their needs. The phases and resources can help to address considerations and recommend quality checkpoints from pre- through post-analytic phases of the total testing process and are intended to be used as resources for method validation from preparation through continual maintenance. The Pathway is based on guidance from the Clinical and Laboratory Standards Institute (CLSI), the College of American Pathologists (CAP), and others.
NOTE: Additional resources are included in the References section of Phase 5.

NGS has revolutionized clinical and public health practice, with clinical and public health laboratories relying on it for numerous applications. Despite the availability and continuing development of guidance for establishing performance specifications and supporting test validations for NGS, challenges remain due to the absence of a standardized quality framework. To help address this, the Pathway can be used to support NGS assay validation, testing implementation, and maintenance of the test system(s). This Pathway is based on the twelve Quality System Essentials (QSEs) developed by CLSI and was cross-walked to the Clinical Laboratory Improvement Amendments of 1988 (CLIA) regulations as well as guidelines for NGS developed by professional organizations. The Pathway provides a framework that accommodates the complexities of NGS, integration into clinical and public health workflows, and the need to maintain a reliable platform that delivers high-quality results.

The Pathway provides a step-by-step framework, including suggestions and links to existing guidance, as well as resources and references to specific, relevant regulations for laboratories to consider when preparing for and completing method validations.

The Pathway is divided into five phases:

• Phase 1 – Prepare for Validation
• Phase 2 – Review and Finalize Procedures
• Phase 3 – Perform Validation
• Phase 4 – Post Validation - Train and Authorize Personnel
• Phase 5 – Test and Maintain

The five phases are comprised of multiple sections, with each section comprised of multiple steps. For example, the first phase, “Prepare for Validation,” is comprised of sections for “Personnel Performing Validation,” “Equipment,” “Reagents, Media and Supplies,” and “Laboratory Space and Safety,” with the “Personnel Performing Validation” section including “Job Descriptions and Role-Based Responsibilities,” “Qualification Requirements,” “Personnel Training,” and “Approval of Personnel.” Each step is further broken down into a checklist with brief descriptions, cross-references to relevant standards and regulatory requirements, and relevant published NGS QI resources.

For clinical and public health laboratories, this tool can contribute to the development of NGS workflows in diagnostic, environmental, surveillance, and research settings that include the necessary components of a QMS, documentation of quality control, integration of quality practices for development and maintenance of bioinformatics pipelines, and compliance with CLIA requirements. NOTE: Personnel may use the references pages for more information on the guidance provided.

Disclaimer:
This tool is not inclusive of all existing guidance and resources currently available, but rather incorporates relevant published NGS QI resources that are agnostic and customizable for laboratories to adopt for their intended use.