Suzanne de Bruijn

120 Chapter 3.1 SUPPLEMENTARY MATERIALS AND METHODS DNA sequencing and variant identification Genomic DNA was isolated from peripheral blood lymphocytes following standard procedures. Subsequently, exome enrichment was performed using the Agilent SureSelect Human All Exome V5 kit according to the manufacturer’s protocols. Exome sequencing was performed on an Illumina HiSeq system by BGI Europe (Copenhagen, Denmark). Readmapping along the hg19 reference genome (GRCh37/hg19) and variant calling were performed using BWA V.0.78 1 and GATK HaplotypeCaller V.3.3 2 . A coverage of >20 reads was reached for 85.1% to 97.8% of the enriched regions. For variant annotation an in-house developed annotation and variant evaluation pipeline was used. For sequencing data of familyW97-056, copy number variant (CNV) detection was performed using CoNIFER V.0.2.2. 3 Genome sequencing was performed by BGI (Hong Kong, China) on a BGISeq500 using a 2x 100 bp paired end module, with a minimal median coverage of 30-fold per genome. Structural variants were called using Manta V.1.1.0 4 and CNVs using Control-FREEC. 5 Variants were validated and visualized using the IGV Software (V.2.4). 6 In the index case of family W08-1421, targeted DNA sequencing was performed using MIP sequencing. 7 MIPs were designed covering exons and exon-intron boundaries of a panel of 89 HL genes ( Table S6 ). Sequencing and data analysis were performed as previously described. 8 For each targeted region, an average coverage of 420 reads was obtained. A coverage of >20 reads was reached for 85.4% of the MIPs. Only those called variants were considered that had a quality-by-depth >200 and that were present in less than 10% of the samples that were analyzed in the same sequence run (n=150). VNTR marker analysis Genotyping of Variable Number of Tandem Repeats (VNTR) markers was performed by genomic DNA amplification using touchdown PCR and analysis on an ABI Prism 3730 Genetic Analyzer (Applied Biosystems). Genomic positions of markers were determined using the UCSC genome browser (human genome assembly GRCh37/hg19). Alleles were assigned with the GeneMarker software (V.2.6.7, SoftGenetics) according to the manufacturer’s protocol. Audiometric cluster analysis A k-means clustering algorithm was applied on the last audiogram of affected subjects with the RIPOR2 variant. 9 Each audiogram was first normalized by subtracting the average hearing threshold across all frequencies fromthe data, preventing the algorithm