A Rigorous Interlaboratory Examination of the Need to Confirm NGS-Detected Variants with an Orthogonal Method in Clinical Genetic Testing

Abstract

Orthogonal confirmation of NGS-detected germline variants has been standard practice, although published studies have suggested that confirmation of the highest quality calls may not always be necessary. The key question is how laboratories can establish criteria that consistently identify those NGS calls that require confirmation. Most prior studies addressing this question have limitations: These studies are generally small, omit statistical justification, and explore limited aspects of the underlying data. The rigorous definition of criteria that separate high-accuracy NGS calls from those that may or may not be true remains a critical issue.We analyzed five reference samples and over 80,000 patient specimens from two laboratories. We examined quality metrics for approximately 200,000 NGS calls with orthogonal data, including 1662 false positives. A classification algorithm used these data to identify a battery of criteria that flag 100% of false positives as requiring confirmation (CI lower bound: 98.5–99.8% depending on variant type) while minimizing the number of flagged true positives. These criteria identify false positives that the previously published criteria miss. Sampling analysis showed that smaller datasets resulted in less effective criteria.Our methodology for determining test and laboratory-specific criteria can be generalized into a practical approach that can be used by many laboratories to help reduce the cost and time burden of confirmation without impacting clinical accuracy.