Suzanne de Bruijn

237 Exploring the missing heritability in subjects with hearing loss and EVA SNP genotyping by Sanger sequencing was performed to confirm the presence of the twelve SNPs that are located within the haplotype. 22 SNP-phasing was performed if DNA samples of family members was available. Optical genome mapping Optical genome mapping (Bionano Genomics) was performed as previously described. 45,46 Ultra-high molecular weight DNA was isolated from whole peripheral blood (collected in EDTA tubes) using the SP Blood & Cell Culture DNA Isolation Kit (Bionano Genomics). CTTAAG labeling was performed using the DLS (Direct Label and Stain) DNA Labeling Kit (Bionano Genomics) and the labeled sample was analyzed using a 3x 1,300 Gb Saphyr chip (G2.3) on a Saphyr instrument (Bionano Genomics). An effective coverage of 124x was reached, with a label density of 14.63/100 kb and an average N50 of 279 kb. De novo assembly (using GRCh37 and GRCh38) and variant annotation were performed using Bionano Solve version 3.4, which includes two separate algorithms for SV and CNV detection. Annotated variants were filtered for rare events as described previously. 45 In addition, the genomic region spanning the CEVA haplotype was analyzed visually in Bionano Access version 1.4.3. PacBio long-read sequencing Genomic DNAwas isolated fromperipheral blood according to standard procedures and subjectedto long-readgenomeHi-Fi sequencingusingtheSMRTsequencingtechnology (Pacific Biosciences). Library preparation was performed using the SMRTbell TM Template Prep Kit 2.0 (Pacific Biosciences) following manufacturer’s instructions. Size selection was performed using a BluePippin DNA size selection system (target fragments ~15- 18 kb). Sequence primer V2 and polymerase 2.0 were used for binding. Subsequently, the SMRTbell library was loaded on an 8M SMRTcell and sequencing was performed on a Sequel II system (Pacific Biosciences). Circular consensus sequencing (CCS), Hi-Fi reads, were generated using the CCS (v4.2.0) tool and were aligned to the GRCh37/ hg19 reference genome with pbmm2 (v.1.3.0). The unique molecular yield was 93.46 Gb and the post-alignment Hi-Fi- coverage was 12x (Mosdepth v0.3.1 47 ). SV calling was performed using PBSV (v2.4.0) and annotation was applied using an in-house SV annotation pipeline.