Suzanne de Bruijn

44 Chapter 1.2 BOX 1 - Single molecule real-time (SMRT) sequencing technique To enable sequencing of single DNA-molecules in real-time, two obstacles had to be overcome. First, concentrating the DNA polymerase and its template, SMRT-bell ( Figure 1A ), to the very small observation chambers, which creates a higher signal-to-noise ratio. This problem has been solved by zero-mode waveguide (ZMW) technology, a small hole of approximately 45 nanometer (nm) in diameter. 72 The DNA polymerase with its template is anchored by a strong biotin/ streptavidin interaction to the bottom of the ZMW. Therefore, the laser illumination of incorporating nucleotides is limited to the bottom, which increases the signal-to-noise ratio 63 as ZMW can efficiently distinguish signals of nucleotide incorporation against the background of unincorporated nucleotides ( Figure 1B ). The second obstacle in real-time sequencing of single DNA-molecules was the large size of the fluorescent dye, which interfered with the normal activity of DNA polymerase and caused halting of the enzyme shortly after initiation of DNA synthesis. In the SMRT technology, the dye is attached to the phosphate chain instead of the nucleotide, which is naturally cleaved during DNA synthesis after nucleotide incorporation and results in a single long, natural DNA strand. 63 The real-time sequencing of the circular SMRT-bell is performed in each ZMW that generates continuous long reads ( Figure 1B ). During data processing, the adaptors are removed, and subreads are generated. Subsequently, the combined subreads enable the generation of one highly accurate consensus sequence called the circular consensus sequence (CCS). There is no limit in DNA length that can be sequenced, since it does not require DNA amplification or synthesis. The challenge lies in library preparation, which needs to result in ultra-long dsDNA molecules. 75 The average size of reads is usually >10 kb and for some molecules, it can reach 1 Mb. 59 The main drawback of nanopore sequencing is its relatively high error rate of ~20%. Compared to SMRT technology, in which the error rate can be reduced by high coverage due to CCS, in Nanopore sequencing it is a systematic error and correction can only be achieved by comparison to short-read sequence data. 75 Nevertheless, this technology is rapidly improving to overcome current issues. 71 Application of third generation sequencing in inherited HL and RD Third generation sequencing has revolutionized the field of medical genetics by its superior performance in the analysis of repeated and highly homologous regions, SVs, haplotype phasing, and transcriptome analysis. 76 These technologies are currently mainly used in research applications and show great promise to overcome the disadvantages of SRS methods. In a systematic analysis, Ebbert et al. compared the performance of whole-genome SRS and LRS technologies at repetitive regions in the human genome. Amongst others, they showed that 8.6% of the protein-coding regions of RPGR (associated with X-linked RD) and 12.7% of the protein coding regions of OTOA (associated with HL) are within the unmapped reads of SRS-data, which were resolved by performing LRS. Specifically, they indicated that Nanopore sequencing outperforms PacBio sequencing by resolving 90.4% and 64.4% of the SRS-unmapped regions, respectively. 77