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.