Stepwise sequence analyses

Figure 1 shows the stepwise genetic analyses, starting with Sanger sequencing for two major EYS founder mutations (c.8805C>A; p.Y2935X and c.4957dupA; p.S1653fs) in Japanese patients with RP. Targeted NGS for all coding regions of EYS was conducted by NGS using an Illumina MiSeq system (Illumina Inc., San Diego, CA, USA) for those who were not genetically solved by Sanger sequencing. We finally conducted WGS for those who were not genetically diagnosed by either of the above methods.

Stepwise genetic test protocol. Two frequent founder variants were initially screened in our cohort by Sanger sequencing, then EYS genes were screened using targeted exome sequencing. Whole genome sequencing was applied, then Alu insertions were screened using Sanger sequencing.

We then applied additional Sanger sequencing to detect the Alu element insertion in exon 4 of RP1 in all analyzed patients with RP according to a recent report by Nikopoulos et al.²⁷.