Suzanne de Bruijn

191 Structural variants cause ectopic enhancer-gene contact in retinitis pigmentosa ≤0.0001 in gnomAD. CNVs and SVs were analyzed from WES data using ExomeDepth 9 and WGS data using Canvas Copy Number Variant Caller 10 (Illumina; copy number gain or loss based on read depth) and Manta Structural Variant Caller. 11 For additional unsolved adRP families, or families suspected to harbor RP17-SVs, WES or WGS was performed. Families of Canadian (CA) or South African (SA) origin were analyzed in theNetherlandswith additional families of Dutchorigin.WGSwas performed as described for NL1. For UK families, WGS was executed as described for UK1 or through the NIHR-Bioresource and Genomics England pipelines as previously described. 7,12 Characterization and validation of structural variants Primer sequences and coordinates are listed in Table S2 and PCR conditions for all breakpoint junctions are available upon request. SV breakpoint regions were assessed for presence of microhomology and repetitive elements. Breakpoint regions and junctions were defined as 150 bp flanking sequence surrounding the breakpoint, which were used as input sequences for subsequent analyses. The presence of microhomology at the breakpoints was assessed using multiple sequence alignment between the junction fragment and the 5’ and 3’ breakpoint regions using Clustal Omega. 13 The presence of repetitive elements at the breakpoint regions was assessed using RepeatMasker. 14 To validate the presence of a triplicated region for UK-SV6, a quantitative real-time PCR (qPCR) experiment was performed on genomic DNA from affected individuals from family UK13 (n=2), and unaffected controls (n=2). qPCR was performed using SYBR Green labTAQ Green mix (labTAQ) on a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems). Primer pairs were designed to amplify genes in the suspected triplicated regions and distal and proximal regions on Chr17 outside the triplicated areas as a reference for standard quantity. Primer sequences and chromosomal positions are listed in Table S3 . Each reaction was run in triplicate and was comprised of 2x labTAQ Green mix (labTAQ), 0.8 µl of each primer (10 mM) and 25 ng DNA in a final reaction volume of 20 µl. Cycling conditions were as follows: 95°C for 2 min, followed by 40 cycles at 95°C for 15 s and 60°C for 20s. Dissociation curves were generated by heat denaturation over a temperature gradient from 60-95°C to ensure no primer-dimers had formed and to check for a single amplicon. To verify the presence of a single PCR product, samples were also electrophoresed on a 2% agarose gel. Data were obtained using the QuantStudio TM Real-Time PCR Software (Applied Biosystems) to generate an amplification plot and a