Comprehensive reanalysis of 2,040 undiagnosed clinical whole-genome sequencing cases improves diagnostic yield

Comprehensive genomic analysis, including whole-genome sequencing (WGS), enables iterative data reanalysis, potentially improving diagnostic yield as new gene–disease associations and analytical methods emerge. Although reanalysis has been shown to increase diagnostic yield across disease groups, uncertainty remains regarding its optimal timing and overall impact. We systematically reprocessed and reanalyzed WGS data from a heterogeneous clinical cohort collected over five years to evaluate the effect of reanalysis on diagnostic yield. Clinical WGS cases (~3000) analyzed at the Department of Genomic Medicine, Copenhagen University Hospital, were reviewed. Cases without a causative variant underwent reanalysis. The cohort included singletons and families across major disease groups, including cancer, neurodevelopmental, hematological, immunological, and endocrine disorders. Samples were reprocessed using the latest in-house germline WGS pipeline, including structural variant analysis, and reinterpreted using updated gene panels and latest disease knowledge. Baseline diagnostic yield was 15% in singletons ( n = 2209) and 30% in family probands ( n = 245). Reanalysis increased yield to 19% and 36%, respectively. Of 1551 curated variants, 79 were clinically reportable, predominantly identified through expanded in silico gene panels. Reanalysis increased overall diagnostic yield from 17% to 21% (22–26% excluding sporadic cancers), with greatest benefit from broad gene panels and semi-automated workflows.