1 and family B: II.2 and II.4) at the Center for Human Genome Variation (CHGV) at Duke University, Durham, NC. Prior to sequencing, target regions were captured using the SureSelect

Human All Exon technology (Aligent Technologies). This technology captures consensus coding sequence exonic regions and flanking intronic regions totaling ∼38 Mb of genomic DNA. The resulting short-sequence reads were aligned to the reference genome (NCBI human genome VX-770 mouse assembly build 36; Ensembl core database release 50_361 [Hubbard et al., 2009]) using the Burrows-Wheeler Alignment (BWA) tool (Li and Durbin, 2009). After accounting for PCR duplicates (removed using the Picard software: http://picard.sourceforge.net) and reads that did not align check details to captured regions of the reference genome, the average coverage for these three samples was ∼71× and each sample had >95% of the bases covered. A base within the 37.8 Mb captured

region was defined as covered if ≥5 short reads spanned this nucleotide (Table S10). Genetic differences between each patient genome and the reference genome were identified using the SAMtools variant calling program (Li et al., 2009), which identifies both single-nucleotide variants (SNVs) and small indels. We then used the SequenceVariantAnalyzer software (SVA) (Ge et al., 2011) to annotate all identified variants. SVA was also used to apply quality control filters to the variants identified by SAMtools. High-quality SNVs were obtained using the following criteria: consensus score ≥20, SNP quality score ≥20, and reads supporting SNP ≥3. High-quality indels were obtained using the following criteria: consensus L-NAME HCl score ≥20, indel quality score

≥50, ratio of (reads supporting variant/reads supporting reference): 0.2–5.0, and reads supporting indel ≥3. The exomes of one of the individuals of family C (II.3) and four individuals of family D (II.1, II.2, II.4, and II.5) were captured using the Agilent SureSelect all exon kit V3 (approximately 51.9 Mb of target sequences) and then sequenced in pair ends (2 × 100 bp) on the Illumina HiSeq2000 (v3 chemistry; 3 exomes/lane format) at the McGill University Genome Quebec Innovation Center (Montreal, Canada). The sequences were aligned and the variants were called using GATK (DePristo et al., 2011). After removal of duplicate reads, using Picard, we obtained an average coverage of >80× per target base, with 95% of the target bases being covered at ≥10×. Only variants that meet all the following criteria were considered: base coverage ≥8×, reads supporting the variant ≥3, and ratio of reads supporting variant/reads supporting reference ≥20%. Variants were then annotated using Annovar (Wang et al., 2010). Genotyping of the p.F362V variant in 1,160 controls was performed in the Center for Human Genome Variation at Duke University (Durham, NC).