Suzanne de Bruijn

43 The impact of modern technologies on molecular diagnostics novel sequencing technique called “single molecule real-time” (SMRT) sequencing 60 , and only three years later, Oxford Nanopore technologies introduced nanopore sequencing. 61 Although these two techniques utilize different approaches to sequence genomic DNA, they share two major advantages compared to NGS. First, they are established on PCR-free and real-time sequencing processes, and second, they generate ultra-long sequencing reads, >10 kb. 59,62 These long-read sequencing (LRS) technologies are revolutionizing the genetics field as they provide a further understanding of the normal and morbid anatomy of human genome and thereby can fill the gaps in the molecular diagnostics of genetic diseases. Single molecule real-time (SMRT) sequencing SMRT sequencing relies on ligatinghairpin adapters tobothends of thedouble-stranded template DNA molecule (dsDNA), thereby circulating the dsDNA into the construct called the SMRT-bell. In the next step, primers and DNA polymerase are annealed to the adapter in the SMRT-bell, which will later be utilized for circular consensus sequencing (CCS) ( Box 1 , Figure 1 ). The CCS approach can obtain approximately 83% accuracy (10x coverage on average) with a 13-15% error rate dominated by small insertions and deletions. 62,63 This can be improved to 99% accuracy by selectively sequencing a targeted regionwith an increased coverage of 15x. 64-66 The SMRT technology is a PCR-free approach and requires minimal amounts of reagents and a simple library preparation procedure by which ultra-long dsDNA can be obtained. This technology can provide the result within hours compared to several days for previous approaches. 60 An average read length of 10-15 kb can be reached, which allows de novo assembly, phasing of variants and haplotyping and the detection of large SVs throughout the genome. 59,67,68 Nanopore sequencing Nanopore sequencing is an advanced third generation sequencing technique that offers straightforward sample preparation, requiring minimal reagents or amplification processes. 69 This technology relies on transferring a DNA molecule through a pore and directly detecting eachnucleotide by its effect on an electric current ( Box2 , Figure2 ). 70,71