To facilitate early deployment of whole-genome sequencing (WGS) for severely ill children, a standardized pipeline for WGS analysis with timely turnaround and primary care pediatric uptake is needed. We developed a bioinformatics pipeline for comprehensive gene-agnostic trio WGS analysis of children suspected of having an undiagnosed monogenic disease that included detection and interpretation of primary genetic mechanisms of disease, including SNVs/indels, CNVs/SVs, uniparental disomy (UPD), imprinted genes, short tandem repeat expansions, mobile element insertions, SMN1/2 copy number calling, and mitochondrial genome variants. We assessed primary care practitioner experience and competence in a large cohort of 521 families (comprising 90% WGS trios). Children were identified by primary practitioners for recruitment, and we used the UK index of multiple deprivation to confirm lack of patient socio-economic status ascertainment bias. Of the 521 children sequenced, 176 (34%) received molecular diagnoses, with rates as high as 45% for neurology clinics. Twenty-three of the diagnosed cases (13%) required bespoke methods beyond routine SNV/CNV analysis. In our multidisciplinary clinician user experience assessment, both pediatricians and clinical geneticists expressed strong support for rapid WGS early in the care pathway, but requested further training in determining patient selection, consenting, and variant interpretation. Rapid trio WGS provides an efficacious single-pass screening test for children when deployed by primary practitioners in clinical settings that carry high a priori risk for rare pediatric disease presentations.