Suzanne de Bruijn

42 Chapter 1.2 patient’s age due to increased exposure to damaging environmental factors during life. In line with this observation, there are several reports of a negative correlation between diagnostic yield and age of onset of HL. 3,47 Despite the successes of WES in clinical settings, this technology is inaccurate in detecting SVs, such as a deletion of a single exon, and does not allow variant detection in deep-intronic regions or regulatory elements. Therefore, WGS may be preferred as it provides a more evenly distributed and uniform read coverage, and it is capable of detecting different types of variants across the entire genome. 53-56 In 2017, Carss et al. investigated a large cohort of RD patients, in which WGS was performed for 605 cases, WES for 72 cases, and for 45 cases both technologies were performed. 56 They identified disease-causing variants in 56% of all individuals (404/722), while by using WES alone the diagnostic yield was calculated to be 50%. Subsequently, 45/58 cases that remained unexplained by WES underwent WGS, and pathogenic variants were identified in 14 cases. The authors concluded that WGS has great power to detect pathogenic SVs, variants in non-coding and regulatory regions, and variants in GC-rich regions. The application of WGS revealed the pathogenic variants in 31% of the cases that remained unsolved after WES. These variants were missed mainly due to the poor quality of reads or the incapability of WES to identify SVs. 56 The prices for WGS keep decreasing 57 and the importance of the non-coding regions of the genome has become more evident. Therefore, a shift from exome to genome sequencing will be observed in clinical diagnostics in the near future to overcome the diagnostic gap observed in the application of WES. In 2020, Méjécase et al. provided a practical and cost-effective guideline for current and future genetic testing of RDs in which they proposed to utilize WES or targeted NGS for the initial screening of exons and flanking intronic regions of (candidate- or known RD) genes, reserving WGS solely for cases that remained unresolved. 58 Although NGS techniques have revolutionized the field of medical genetics, these short- read sequencing (SRS) approaches pose several limitations, such as (1) difficulties in the identification of complex and large SVs, (2) inability to sequence repetitive regions, (3) the lack of phasing of alleles, and (4) difficulties distinguishing highly homologous regions such as pseudogenes. 59 These limitations may play a significant role in the diagnostic gap in medical genetics. Third generation sequencing Due to the limitations of the aforementioned NGS techniques, there has been a need to develop new sequencing approaches to overcome these issues. The era of third generation sequencing arrived in 2011, when Pacific Biosciences (PacBio) released a