Exome Capture and Sequencing Protocol

Genomic DNA extracted from fresh blood cells from the proband and her father with QiAmp DNA mini kit (Qiagen) was captured using Agilent in-solution enrichment methodology with their biotinylated oligonucleotides probes library (SureSelect Clinical Research Exome V2, Agilent Technologies), followed by paired-end 75 bases massively parallel sequencing on Illumina HiSeq4000 (IntegraGen SA, Evry, France), as reported (13 (link)). Sequence capture, enrichment and elution were performed according to manufacturer’s instruction and protocols (SureSelect, Agilent) without modification, except for library preparation performed with NEBNext® Ultra kit (New England Biolabs). For library preparation, 600 ng of each genomic DNA were fragmented by sonication and purified to yield fragments of 150–200 bp. Paired-end adaptor oligonucleotides were ligated on repaired fragments then purified and enriched by 8 PCR cycles. A total of 1200ng of the purified Libraries were then hybridized to the SureSelect oligo probe capture library for 72 h. After hybridization and washing, the eluted fraction was PCR-amplified, purified and quantified by qPCR. Each eluted-enriched DNA library was then sequenced on an Illumina HiSeq4000 as paired-end 75 bp reads. Image analysis and base calling were performed using Illumina Real Time Analysis (2.7.7) with default parameters.