Evaluating Genome Sequencing Strategies: Trio, Singleton, and Standard Testing in Rare Disease Diagnosis

PurposeShort-read genome sequencing (GS) is a comprehensive genetic testing method capable of detecting multiple variant types. Despite its technical advantages, systemic comparisons of singleton GS (sGS), trio GS (tGS), and exome sequencing-based standard-of-care (SoC) in real-world diagnostics remain limited. MethodsWe systematically compared sGS, tGS, and SoC genetic testing in 448 patients with rare diseases in a blinded, prospective study. Three independent teams evaluated the diagnostic yield, variant detection capabilities, and clinical feasibility of GS as a first-tier test. Diagnostic yield was assessed through both prospective and retrospective analyses. ResultsIn prospective analyses, tGS achieved the highest diagnostic yield for likely pathogenic/pathogenic variants (36.8%) in a newly trained team, outperforming the experienced SoC team (36.0%) and the sGS team (30.4%). Retrospective analyses, accounting for technical variant detection and team experience differences, reported diagnostic yields of 38.6% for SoC, 41.3% for sGS, and 42.2% for tGS. GS excelled in identifying deep intronic, non-coding, and small copy-number variants missed by SoC. Notably, tGS additionally identified three de novo variants classified as likely pathogenic based on recent GeneMatcher collaborations and newly published gene-disease association studies. ConclusionGS, particularly tGS, demonstrated superior diagnostic performance, supporting its use as a first-tier genetic test. sGS offers a cost-effective alternative, enabling faster, more efficient diagnoses for rare disease patients.