Operationalizing accreditation standards for laboratory-developed next-generation sequencing validation of CFTR amplicon-based assays.

INTRODUCTION: Application of College of American Pathologists (CAP) standards for molecular next-generation sequencing assays, typically high-complexity laboratory-developed tests, requires assay-specific, principled translation. We sought to systematically apply CAP laboratory-developed test validation requirements for next-generating sequencing to targeted CFTR gene sequencing on an Illumina iSeq100 Sequencing System and to address assay-specific translational guidance gaps. METHODS: We evaluated the analytic performance (accuracy, precision, analytic sensitivity, and specificity) and other requirements (eg, confirmatory testing, proficiency testing) in a CAP-accredited laboratory, including assessment of logistics (eg, turnaround time, cost), with particular attention to feasibility for smaller laboratories. RESULTS: Variant calls showed complete concordance across analytic parameters but, as the sole outcome measure, obscured subthreshold effects of experimental variables and random variability. Critical site depth analysis was essential to reveal these effects. DISCUSSION: Per-sample costs depend on batch size; smaller laboratories can reduce costs through centralization or sample pooling, although pooling requires longer turnaround (weeks to months), even on a iSeq100 system, potentially causing misalignment of CAP proficiency testing and batch schedules. This issue mandates self-evaluation and code 11 selection for CAP proficiency testing submissions. Real-time sample preparation, however, precludes running negative and internal controls with each processing run, necessitating process controls (eg, minor allele frequency, alternate allele determination) and an individualized quality control plan.