Targeted NGS for Inherited Retinal Diseases

Genomic DNA was extracted from peripheral blood of all family members with the TIANGEN Blood DNA Kit (DP304, TIANGEN, China). Targeted next generation sequencing (NGS) was performed for the proband using a custom designed panel (PS400) containing 376 known genes associated with inherited retinal diseases (Zhu et al., 2021 (link)). The Nextseq 500 (Illumina, San Diego, CA, USA) platform was used for paired-end sequencing with read lengths of 150 bp and average sequencing depth of almost 300 X. Raw reads were mapped to the human genome reference (UCSC hg19) using three commercial software including XYGeneRanger 2.0 (Xunyin, Shanghai, China), TGex (LifeMap Sciences, Alameda, CA, USA) and Efficient Genosome Intepration System, EGIS (SierraVast Bio-Medical Technology Co., Ltd, Shanghai, China). Variant-filtering was based on public and in-house SNP databases, including 1000Genome project, HGMD, ExAC and ClinVar, as well as our internal database. The non-synonymous and splicing variants with MAF <2% were kept for further analysis. Sanger sequencing and co-segregation analysis were performed for the verification of suspicious disease-relevant gene variants in the available family members. Primer sequences and PCR conditions were shown in Supplementary Tables 1, 2, respectively.