Taylor et al. consider the utility of whole-genome sequencing for diagnosis of genetic disorders in routine clinical practice as part of the WGS500 project to sequence the whole genomes of 500 patients. The authors examine whole-genome sequencing data from 217 individuals (including 156 independent cases or families) with a range of genetic disorders for which previous genetic screening had not identified any pathogenic variants. They demonstrate that whole-genome sequencing now allows the identification of at least one variant with a high level of evidence of pathogenicity in 21% of cases (33/156), with higher rates seen for Mendelian disorders (34%; 23/68) or family trios (57%; 8/14). The authors consider analysis strategies that improve the accuracy of variant calling and detection rates, and they review challenges in sequence interpretation and in establishing the pathogenicity of variants.