Suzanne de Bruijn

61 The impact of modern technologies on molecular diagnostics complex than initially thought. A quantitative (gene expression levels) or qualitative (isoform structures, novel exons) analysis of the transcriptomic landscape is valuable in enhancing diagnostic yield, as shown by several studies. 195,196 In combination with genome sequencing, RNA sequencing can improve the interpretation of variants with unknown significance, although inaccessibility of cell types for RD and HL-associated genes is a major limitation. LRS offers the potential for RNA analysis as well: for example, the Iso-Seq method of PacBio enables the sequencing of full transcripts and Nanopore offers the direct sequencing of RNA molecules. 59 LRS techniques have already shown to be successful in the identification of novel full-length transcripts. In a study performed by Ray et al., an abundant retina-specific CRB1 transcript ( CRB1-B )was detectedwhichwas not annotated in genome databases, such as the UCSC genome browser. 164,197 The authors showed that the expression of the CRB1-B transcript is significantly higher in photoreceptors than the canonical CRB1 transcript ( CRB1-A ). The newly identified transcript includes unique exons that are not present in CRB1-A and thereby represent important candidate regions for potentially missed pathogenic variants. 197 In addition, developments in the single- cell RNA sequencing field allow the identification and characterization of tissue-specific isoforms and regulatory events. The Single Cell Portal (Broad Institute) offers a valuable resource of tissue-specific single-cell RNA sequencing datasets. 198 Epigenomics is an emerging andpromisingdevelopment in the fieldofmedical genetics. Analysis of epigenomic signatures can aid in the understanding of the 3D organization of the genome. Since base-modifications remain captured in native DNA molecules that are used for SMRT and Nanopore sequencing, investigation of the methylome and DNA base modification is possible. 59,62 Ideally, multi-omics layers (e.g. genomics, transcriptomics and epigenomics) should be integrated (so called multi-omics), which aids in an ultimate understanding of the genomic landscape and provides valuable insights for (candidate) disease-associated genes. CONCLUSIONS AND DISCUSSION Fifty years after the arrival of the Sanger sequencing technique, the sequencing technology landscape is still rapidly evolving. However, genetic diagnostic yield still varies between 40-70% for inherited HL and RD, indicating that there are still opportunities for further improvement. 3,4,47 Although novel disease-associated genes are being discovered, disease-gene identification curves are slowly reaching a plateau phase, suggesting more attention should be paid to currently missed or