Exome sequencing, variant calling and Sanger sequencing
Exomes sequencing (ES) was performed on the female proband and her parents in order to identify the causative mutation. The ES protocol achieved a mean coverage of 200× over 98% of the targeted regions. A minimum coverage of 20× was the standard (AmCare Genomics Laboratory, Guangdong, China) in the regions that contained 5,177 disease candidate genes. The Speedseq Toolkit was used for the data analysis by: 1) performing the human genome alignment with BWA-MEM, 2) marking duplicates using SAMBLASTER, 3) sorting and performing indexing of BAM files with Sambamba, and 4) executing variant calling using FreeBayes. Speedseq is an ultra-fast personal genome/exome analysis toolkit for next generation sequencing(C. Chiang et al., 2015). It takes roughly 2 hours to complete the analysis of one ES sample by using 7 core 14 threads of the CPU. To quickly and accurately detect pathogenic variants, we used EXOMISER to help narrow down the potential candidate variants. Variants were prioritized according to pathogenicity, quality, inheritance pattern, model organism phenotype data and phenotypes known to be associated with disease genes in humans(Smedley et al., 2015). Sanger sequencing was used to determine the segregation across all family members. The ARR4 c.569C>G (p.S190*) primers for PCR amplification were: 5’-CCATAGGAAAACAGGCATCAGG-3’ (forward) and 5’-CCTTGTTGGTGCAGTTGTTG-3’ (reverse).
Cell culture and transfection
The APRE19 human retinal pigment epithelial cell line was used in the study. DAPI staining was performed to detect whether cells were contaminated with mycoplasma. Cell culture was performed using DMEM containing 10% fetal calf serum at 37°C with 5% CO2.