Trusight one kit

DNA samples were extracted from peripheral blood using a QIAmp DNA Blood Mini Kit (Qiagen, Germany). Libraries were prepared using TruSight One kit (Illumina, USA) in Family 1 and SureSelect Human All exon V2 kit (Agilent Technologies, USA) in Family 2 following manufacturer’s instructions. Paired-end sequencing was performed on an Illumina Genome Analyzer II platform (Illumina, USA). Reads were mapped against the human reference genome hg19 using the BWA software and analyzed with the GATK Unified Genotyper v2.8. Amplicon-based deep sequencing of specific exons of the PLCG2 gene (RefSeq NM_002661.3) was performed as previously described to evaluate parental gene mosaicism [10 (link)]. For Sanger sequencing, specific exons of the PLCG2 gene were amplified by in house–designed PCR (primers listed in Supplementary Table S2), purified with Illustra ExoStar 1-Step kit (GE Healthcare, USA), bidirectional sequenced using ABI BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, USA) and run on an automated ABI 3730XL analyzer (Applied Biosystems, USA).

All procedures were conducted as part of routine clinical care. The study was performed under the ethics guidelines issued by our institution, with written informed consent obtained from all participants for genetic studies.
Genetic analysis for PP genes SCN4A, CACNA1S, and KCNJ2 was performed at the Neurogenetics Unit, National Hospital for Neurology and Neurosurgery as provided by the Channelopathy Highly Specialized National Service for rare disease. Samples underwent next-generation sequencing on an Illumina HiSeq after enrichment with an Illumina custom Nextera Rapid Capture panel (Illumina, Inc, San Diego, CA). For case 2, the library preparation and enrichment were performed with TruSight One kit (Illumina) according to protocol instructions, allowing analysis of a panel of ≈5,000 genes (including PP genes and RYR1). The library was quantified with the Qubit 2.0 Fluorometer system (Thermo Fisher Scientific, Waltham, MA), and 2 × 250-bp paired-end sequencing was performed on the MiSeq sequencer (Illumina), as well as sequences alignment (Burrows-Wheeler aligner) and variant calling (Genome Analysis Toolkit variant caller). The variants were then analyzed with VariantStudio (Illumina).
Additional targeted RYR1 Sanger sequencing of all cases was performed at the Diagnostic DNA Laboratory at Guy's Hospital, London.

The sequencing was performed using the TruSight One Kit (Illumina, San Diego, CA, USA), which targets 4800 genes associated with human pathology (12 Mb). This included around 150 genes or candidate genes associated with the clinical phenotype for DSD. The sequencing was performed with the MiSeq platform (Illumina, San Diego, CA, USA) [16 ] using the manufacturer’s instructions. Bioinformatic analysis was performed using Galaxy bioinformatic platform, and variant interpretation was based on ACMG recommendations [17 (link)].