Sureselect all exon v4

In this study, 269 cCSC patients who visited the outpatient clinic of the Department of Ophthalmology of the Radboud university medical center, Nijmegen, the Netherlands were included. Patients were diagnosed with cCSC based on multimodal retinal imaging as described previously^{6 (link),10} . DNA of cCSC patients, isolated from blood, was purified with the QIAamp DNA purification kit (Qiagen), according to manufacturer’s instructions. 3 µg of purified DNA was used as input for the SureSelect^XT target enrichment system for Illumina paired-end multiplexed sequencing (Version B4, August 2015, Agilent Technologies).
Library preparation was performed according to the manufacturer’s instructions. Samples were hybridized with the SureSelect All Exon V4 (Agilent Technologies) capture library and post-hybridization addition of the index tags was performed on the Dynabeads MyOne Streptavidin T1 beads (ThermoFisher Scientific). Midway and final quality checks were performed with Tapestation high sensitivity D1000 screentape (Agilent Technologies) and a Qubit dsDNA high sensitivity assay (ThermoFisher Scientific). A total of 8 samples was pooled for sequencing in one lane. Sequencing was performed at the Department of Genetics of Maastricht University Medical Center+, Maastricht, The Netherlands, using an Illumina HiSeq2000 with 2*100 bp chemistry.

Genomic DNA was extracted from whole bone marrow aspirate samples using Autopure Extractor (QIAGEN). Exome capture hybrid was performed with using Agilent SureSelect All Exon V4. Illumina HiSeq 2000 sequencer was used for sequencing with 75 base pair paired end read. Sequencing data was aligned to the hg19 human genome reference using Burrows-Wheeler Aligner (BWA) [33 (link)] followed by mark duplication, indel realignment, and base recalibration using Genome Analysis Toolkit (https://www.broadinstitute.org/gatk/guide/best-practices?bpm=DNAseq). The resulting BAM [34 (link)] files were preprocessed and base substitutions and small insertions/deletions were called using Mutect [35 (link)] and Pindel [36 (link)], respectively. To overcome the lack of matched normal sample, we generated virtual common normal sequence using in-house pooled normal sequence. This method has been shown to function as almost equivalent as matched normal to call somatic variants [37 (link)]. Since we intended to use mutation data for potential clinical application, we wanted to be conservative on variant annotation. Therefore, we modified approach used by Pappaemanuil et al. (5) to call high-confidence driver mutations (Supplementary Methods).