Suzanne de Bruijn

48 Chapter 1.2 Despite the advantages of LRS techniques, they possess multiple important drawbacks that prevent a wide range of uses outside research applications. One of these is the relatively high costs compared to SRS NGS technologies ($800-2,000 per run depending on the different platforms and instruments), based on the lowest possible flow cell price and highest output. 62 The other major disadvantage is the requirement for high quality ultra-long dsDNA, which can be challenging to obtain. In particular for Nanopore sequencing, the required fresh blood samples for DNA extraction can also be a hurdle. However, as LRS technologies are rapidly decreasing in price and are continuously improving in different aspects, such as optimized library preparation and error correction, it is expected that these technologies will eventually enter routine genetic diagnostics in Western countries. In addition, like SRS, targeted LRS can also be performed by targeted amplicon sequencing, CRISPR/Cas-based targeted enrichment, or using a “Read Until” approach, in order to enrich for genetic loci associated with a specific phenotype. Targeted LRS is a cost-effective and efficient strategy to investigate high-priority loci in unsolved cases. 81,82 For both HL and RD, several associated genetic loci (44 and 36 loci, respectively) have been described for which the implicated genetic defect is still elusive 1,2 , and therefore, a targeted LRS approach could be of interest. Finally, as sequencing technologies develop and improve rapidly ( Figure 3 ), the next challenge will lie in bioinformatics, data storage, data analysis, and variant interpretation of NGS or LRS data. A high number of different variants are revealed by these methods. However, not all these variants are disease causing. Therefore, special attention is being paid to prioritization processes that can aid in decreasing the number of putative candidate variants. In addition, developments in bioinformatic tools are needed to better interpret the effect of candidate variants. In the next section, we will discuss the importance and challenges of variant interpretation and the importance of this matter in clinical application. VARIANT INTERPRETATION The total length of human DNA is over 3 billion base pairs and holds on average 4-5 million variants compared to the healthy human reference genome, which highlights the obvious challenge of distinguishing potential disease-causing variants from benign variants or polymorphisms. 83 For protein-truncating variants, a potential pathogenic consequence is often evident, whilemissense, synonymous and non-coding variants are more challenging to interpret. Moreover, with the increased knowledge regarding the involvement of the non-coding DNA in human disease development, the complexity of data to be analyzed has gone through the roof.