A stepwise short- and long-read whole genome sequencing strategy resolves previous genetically unresolved inherited retinal disease cases

Purpose A significant proportion of cases with rare inherited retinal disease (IRD) remain genetically unresolved following short-read whole genome sequencing (WGS). This study aimed to increase the diagnostic yield of two cohorts in a research setting. One cohort was previously screened by short-read WGS (n=120 cases), while the second cohort (n=28 cases) was not included in any previous short-read WGS studies. In contrast to the larger cohort, these cases only underwent either exome sequencing (ES) or single-molecule molecular inversion probe (smMIPs) sequencing as pre-screening in earlier studies. For all cases, we performed a stepwise, case by case reanalysis in which short-read WGS was generated (n=28 cases) and (re)analyzed short-read WGS data using an optimized approach. For a subset of cases (n=20) that remained unresolved, this was followed by long read WGS. Together, these steps aimed to improve the diagnostic yield in these cohorts. Methods Short-read WGS data of 148 IRD-cases were (re)analyzed using updated allele frequency databases, new variant caller and variant predictor tools, as well as updated and extended gene-panels to identify causal single nucleotide variants (SNVs) and structural variants (SVs). For 20 genetically unresolved cases with sufficient high-quality DNA available, long-read WGS was performed. Results The (re)analysis of short-read WGS data in the 120 previously WGS-screened unresolved cases resulted in a diagnostic yield of 15% (18/120). For the 28 individuals without prior WGS, performing WGS analysis within this study provided an additional yield of 32% (9/28). Furthermore, long-read WGS contributed two genetic diagnoses among the 20 long-read WGS-sequenced probands. Amongst other reasons, new genetic diagnoses were attributed to pathogenic variants in genes newly associated with IRDs and in genes that were not included in the gene panels applied in previous WGS studies. The Mobile Element Locator Tool facilitated the identification of two pathogenic Alu insertions in short-read WGS. Long-read WGS identified two additional pathogenic variants. Conclusion These results support periodic reanalysis of existing short-read WGS data as a key component of IRD genetic diagnostics, with selective long-read WGS representing a valuable additional approach for unresolved cases. Together, these complementary strategies offer a practical framework for improving molecular diagnosis and narrowing the remaining diagnostic gap in IRDs.