Suzanne de Bruijn

40 Chapter 1.2 Sanger sequencing is still routinely used for variant validation and has an extremely high accuracy of up to 99.999%. 33 However, it is considered a low-throughput technique as up to 1 kilobase (kb) of DNA can be sequenced in 96 or 384 parallel reactions. 34 The technique has been optimized by the application of nucleotide-specific fluorescent labels and automated detection 35,36 , the invention of the polymerase chain reaction (PCR) 37 , and the usage of polyacrylamide gels in capillary electrophoresis. 36 Therefore, DNA sequencing can be achieved within a shorter time frame and on a larger scale, in which the sequencing of millions of reads can be carried out in parallel called “massive parallel sequencing” or “NGS”. The NGS technique has rapidly overcome the limitations of traditional sequencing. Since 2005, various sequencing platforms such as Illumina, Ion Torrent, Roche 454, and SOLiD sequencing have been developed, which has resulted in a rapidly changing landscape during this new era of sequencing. The read length of these different platforms is shorter than that of Sanger sequencing (approximately 50-500 bp) and with a higher error rate (0.1% in NGS compared to 0.001% in Sanger sequencing). 38 However, the fast development of NGS techniques and the generation of public reference datasets containing population allele frequency data allowed a widespread integration of NGS technology in the research community and later in the clinical diagnostics of genetic diseases. Nevertheless, as whole genome sequencing (WGS) is still relatively expensive and data interpretation is complex, a targeted sequencing approach (e.g. whole exome sequencing (WES)) is often preferred. Targeted capture sequencing Genomic regions of interest, such as the genes implicated in HL or RD, can be selectively enriched before sequencing is performed. There are various methods available to enrich for target regions such as hybridization-based, highly multiplexed PCR-based, and targeted circularization approaches. Extensive studies have been performed which have applied these techniques to unravel genetic defects involved in inherited HL and RD. In 2013, Chio et al. investigated 32 cases with familial non-syndromic HL, in which they reached a molecular diagnostic rate of 37% using a candidate gene sequencing approach of GJB2 , SLC26A4 , POU3F4 or mitochondrial genes, based on observed clinical features and inheritance patterns. Later, by application of hybridization-based target capture sequencing for 80 HL-associated genes, they were able to increase the total diagnostic detection rate to 78% in this cohort. 39 In 2017, Dockery et al. utilized the hybridization-based enrichment method to sequence 254 RD-associated genes in over 750 affected individuals in Ireland, in which they could identify pathogenic variants in 68% of the cases. 40 A recent study by Khan et al. applied a highly multiplexed PCR-based approach with single-molecule molecular inversion probes (smMIPs) to sequence