Sequencing statistics. A: Schematic drawing of a 12-plex 2.1M NimbleGen sequence-capture array. Samples were barcoded and hybridized individually. After hybridization, all 12 samples were eluted simultaneously, allowing to proceed with one pooled sample consisting of 12 different DNAs. B: Histogram of median target coverage. Only 15 targets were covered less than five times. Most targets show a coverage of 20–30×. C: The minimum coverage of a percentage of targets. Solid line indicates the average across 100 samples, whereas dotted and dash lines indicate first and second standard deviation respectively. D: Evenness of coverage for all samples. The average evenness score over all samples is 97.3%. E: Example of a poorly covered target. This screenshot shows the coverage of some exons of GRM6. The box highlights exon 1, which is poorly covered in comparison with the rest of the gene. F: The 15 targets that are poorly covered (median coverage of less than five times) have either a high GC content (often an UTR or exon 1) or are containing repeat-rich regions.

Systematic variant prioritization tool to efficiently reduce the number of identified variants. A: Filtering of identified variants in the 12 samples, each carrying two variants in recessive RD genes. 14,144 genetic variants were automatically detected, including 21 of the 24 known mutations. A further systematic prioritization (described in Material and Methods) reduced the number of variants to 97, including 18 of the 24 known pathogenic mutations (step 2). Of these 97 variants, all variants were selected that were consistent with the known inheritance pattern of the respective gene, resulting in 44 remaining variants, including 16 of the known variants (step 3). A manual search for a variant on the second allele was performed for recessive genes where only one variant was found (step 4), resulting in a total number of 48 variants, now including 20 of the 24 known mutations (step 5). B: In the group of 100 RP samples with unknown cause of disease, in total 128,557 variants (including six larger deletions) were automatically annotated (step 1). After applying the same filtering steps as for the 12 known RD samples, 359 variants remained after prioritization (step 5). RD, retinal dystrophy.

De novo mutations in isolated RP patients. A: De novo mutation in PRPF31. In patient 27,790, a heterozygous nonsense mutation was detected in the autosomal dominant RP gene PRPF31 that was not present in both the unaffected parents. To confirm that individuals DNA11-01005 and DNA11-00996 are the biological parents, 16 highly polymorphic markers distributed across the genome were analyzed, showing a perfect Mendelian inheritance for all markers, thereby confirming the de novo event (Supp. Fig. S4). B: De novo mutation in RHO. In patient 22,315, a heterozygous missense mutation was detected that was predicted to be pathogenic (indicated in red). The mutation was not present in three unaffected siblings and the unaffected mother. Because the unaffected father was deceased, seven microsatellite markers surrounding and in close proximity of RHO were analyzed to determine haplotypes and the likelihood of a de novo event. The proband 22,315 and his three siblings all appeared to have inherited the same RHO allele from the deceased father, strongly suggesting that the RHO mutation has occurred by a de novo event. The genomic position of the microsatellite markers and RHO is indicated between parentheses. C: De novo mutation in USH2A. In patient 28,557, compound heterozygous mutations were detected in the autosomal recessive RP gene USH2A. One mutation (M1) was inherited from the mother, but the second mutation (M2) was not present in both parents, and as such, had occurred by a de novo event. Again, parental testing confirmed that DNA11-02253 and DNA11-02247 were the biological parents of the patient (Supp. Fig. S4).