Whole Exome Sequencing Variant Filtering

Patient 1 was detected in a family-based WES study (unpublished data (CH, GV, Filip Van Nieuwerburg, NVdA, RFK)). Patient DNA was fragmented using Covaris^® M220 Focused-ultrasonicator™ (Covaris, MA, USA), followed by TruSeq DNA Sample Preparation (Illumina Inc, San Diego, CA, USA), enrichment using the SeqCap EZ Human Exome Library v3.0 kit (NimbleGen, Roche, Penzberg, Germany), and sequencing on HiSeq 2000 (Illumina Inc, San Diego, CA, USA), all following standard protocols. Data analysis was performed using Galaxy (see URLs)^{49 (link)-51 (link)}. Variants were filtered by VariantDB (see URLs, manuscript in preparation) to exclude variants with (1) low quality, using thresholds based on correlation between NGS data and SNP-chip genotyping; (2) intronic or intergenic location, except splice sites; and (3) inheritance from the parents. WES sequencing of patients 2, 3 and 4 was performed as described^{2 (link),8 (link)}. The mutation in patient 5 was identified in a family trio based study. WES was performed using Illumina technology (Illumina Inc, San Diego, CA, USA), and sequence data was returned and analysed using software supplied from Oxford Gene Technology. Presence of reported (de novo) mutations were confirmed by an independent technique such as Sanger sequencing. Raw sequence data will be uploaded in The European Genome-phenome Archive (EMBL-EBI) database.