Suzanne de Bruijn

46 Chapter 1.2 BOX 2 - Nanopore sequencing technique Nanopore sequencing occurs in a flow cell, in which two ionic solution compartments are separated by a membrane containing thousands of nanopores. The flow of electric current between these compartments depends on the molecule transferring through one of the pores. Since each nucleotide differs in shape, its effect on electric current is specific for each nucleotide. 62,71,73 Library preparation in Nanopore sequencing includes end-repair of the ultra-long dsDNA, dA-tails addition, size selection and ligation of adapters (protein-DNA molecules). The first adapter is the leader-adapter, which contains a motor enzyme. It binds to the nanopore and ensures gradual movement of DNA through the pore. The dsDNA is then unwound at the pore and only one strand will pass through the pore. The second adapter is a hairpin-adapter containing a hairpin protein. It generates one long single-strand of DNA, which ensures the sequencing of the second strand of DNA in order to increase sequencing accuracy. 61,62,74 One important application has been to identify complex SVs associated with genetic diseases including HL and RD. Reiner et al. utilized SMRT LRS to detect a 72.8-kb deletion region in the BBS9 gene and map the breakpoints at the nucleotide level in a patient diagnosed with Bardet-Biedl syndrome. This deletion was determined to be the causal variant and a founder mutation in the Guyanese population. 78 In another recent study, researchers utilized transcriptome sequencing followed by short- and long-read WGS to identify a 7.4-kb duplication in NMNAT1 , which spans two out of five exons of this gene. The duplication caused a previously unrecognized autosomal recessive syndrome, symptoms of which are Leber congenital amaurosis and sensorineural HL, which occur together with other features such as spondylo- epiphyseal dysplasia, intellectual disability, and brain anomalies. The authors were able to determine the exact breakpoints of the duplicated region, missed by previous approaches, as well as two Alu elements flanking this segment which are potentially involved in the origin of the SV. 79 Recently, Nanopore sequencing enabled researchers to unravel the genetic defect in two unrelated patients diagnosed with mild-to-moderate HL. Nanopore sequencing revealed a gene conversion event between the OTOA gene and its pseudogene, in which exons 21 to 23 of OTOA were replaced by exons 1 to 3 of OTOAP1 . 80 As pathogenic variants within the OTOA gene have been described to cause autosomal recessive HL (DFNB22), this gene conversion event was considered causative. 80