To rule out variants that arose from spontaneous cell line artifacts, somatic mosaic mutations, or sequencing errors, we validated all homozygous variants in all family members using Sequenom technology. Genotyping candidate variants in the 16 probands allowed us to examine inheritance of variants as well as segregation with disease, since many families had multiple affected individuals as well as unaffected siblings (Figure S2). Variants that did not validate with Sequenom genotyping despite high sequencing depth (≥100) generally occurred in regions of the genome that were not uniquely mappable. For uniquely mapped variants, the rate of validation correlated well with sequencing depth (Pearson's correlation = 0.532, P = 0.001×10−30, t-test) (Figure S3). Analysis of segregation further permitted us to focus on bona fide inherited mutations as we only considered those variants that were homozygous in the proband (by whole exome sequencing and Sequenom confirmation), heterozygous or absent in unaffected siblings, and transmitted from heterozygous parents. This validation step thus eliminates any possible sequencing errors or somatic mutations that complicate many high-throughput sequencing studies. We overlaid the validated variants with the result
