Pcr primers

A total of 135 inherited retinal degeneration-related genes (Supplementary Table 1) were selected using a solution-based sequence capture custom enrichment kit (GenCap, MyGenostics, Beijing, China). Peripheral blood samples were collected from the proband, and genomic DNA extraction was performed using a QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA, USA). The quantity and quality of the DNA were verified using a NanoDrop 2000 (Thermo Fisher Scientific, Delaware, USA). Next, genomic DNA (1 μg) was fragmented into 150-300 bp by a Covaris sonicator (Covaris S2, Massachusetts, USA). After end-repair and adapter ligation, the fragments underwent 4 cycles of PCR amplification with Illumina PCR primers. The purified PCR products (500 ng) were hybridized to the GenCapTM probe (in solution) for 16 hours. After hybridization, washing, and elution, the eluted fraction underwent 14 cycles of PCR amplification and was subsequently subjected to Qubit and quantitative PCR to estimate the magnitude of enrichment. Each eluted, enriched DNA sample was then sequenced on HiSeq2000 Analyzers (Illumina, San Diego, USA) for 90-bp paired-read sequencing, providing an average coverage depth of each sample of at least 100-fold. Image analysis and base calling was performed using Illumina Pipeline (version 1.3.4) with default parameters.

hmeDIP-Seq was performed as previously described [61 (link)]. Briefly, DNA was sonicated to yield fragmented DNA with a modal size of 150 bp. Following end-repair and A-tailing, Illumina TruSeq adaptors were added to the resulting DNA according to the manufacturer’s instructions. The adaptor ligated DNA was then subjected to hMeDIP as outlined above. The resulting input and hMeDIP samples were PCR amplified for 15 cycles using single-end Illumina PCR primers. Finally, libraries were size selected by agarose gel electrophoresis. All DNA purification steps were carried out using Agencourt AMPure Beads (Beckman Coulter, Brea, California, USA), and DNA quality, quantity and size were tested after every step on an Agilent Bioanalyser using High Sensitivity DNA chips (Agilent Technologies, Santa Clara, California, USA Samples were subjected to single-end sequencing on the Illumina GAIIx platform. Sequencing was carried out at the Genomics Core, Albert Einstein College of Medicine, New York.