Clinical, Rapid Whole-Genome and -Exome Sequencing, Analysis, and Interpretation

Clinical urWGS, rWGS, and rWES were performed in laboratories accredited by the College of American Pathologists and certified through the Clinical Laboratory Improvement Amendments. Each step included benchmarked quality assessment. Experts selected a few clinical features representative of each child’s illness from the Electronic Health Record (Epic) and mapped them to simple genetic diseases with VAAST.³² Trio EDTA-blood samples were obtained where possible and all samples were sequenced upon receipt. Genomic DNA was isolated with an EZ1 Advanced XL robot and the EZ1 DSP DNA Blood kit (QIAGEN). DNA quality was assessed with the Quant-iT Picogreen dsDNA assay kit (ThermoFisher Scientific) using the Gemini EM Microplate Reader (Molecular Devices). Genomic DNA was fragmented by sonication (Covaris), and bar-coded, paired-end, PCR-free libraries were prepared for rWGS with TruSeq DNA LT kits (Illumina) or Hyper kits (KAPA Biosystems). Sequencing libraries were analyzed with a Library Quantification Kit (KAPA Biosystems) and High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical), respectively. 2 × 101 nucleotide rWGS and urWGS was performed to at least 40-fold coverage with Illumina HiSeq 2500 (rapid run mode), HiSeq 4000, or NovaSeq 6000 (S1 or S2 flow cell) instruments, as described.¹²

Sample preparation and sequencing for rWES was performed by an external clinical laboratory (GeneDx). Exome enrichment was with the xGen Exome Research Panel v1.0 (Integrated DNA Technologies), and amplification used the Herculase II Fusion polymerase (Agilent).³³ FASTQ files for rWES were transferred to Rady Children’s Institute for Genomic Medicine (RCIGM) for analysis and interpretation.

urWGS, rWGS, and rWES sequences were aligned to human genome assembly GRCh37 (hg19), and variants were identified with the Illumina DRAGEN (Dynamic Read Analysis for GENomics) Bio-IT Platform (v.2.5.1, Illumina; Table S2).¹² Structural variants were identified with Manta and CNVnator (using DNAnexus), a combination that provided the highest sensitivity and precision.¹² Structural variants were filtered to retain those affecting coding regions of known disease-associated genes and with allele frequencies < 2% in the RCIGM database. Nucleotide and structural variants were annotated, analyzed, and interpreted by clinical molecular geneticists using Opal Clinical (Fabric Genomics), according to standard guidelines.³⁴ Interpretation was initially performed with proband sequences alone, to determine the diagnostic yield of singleton sequencing (Figure 1). If no diagnosis was forthcoming, interpretation was performed again, with parental samples if available, in order to determine the net increase in diagnostic yield of duo or trio sequencing (Figure 1). Opal annotated variants with respect to pathogenicity, generated a rank ordered differential diagnosis based on the disease gene algorithm VAAST, a gene burden test, and the algorithm PHEVOR (Phenotype Driven Variant Ontological Re-ranking), which combined the observed HPO phenotype terms from infants, and re-ranked disease genes based on the phenotypic match and the gene score.¹² Automatically generated, ranked results were manual interpreted through iterative Opal searches. Initially, variants were filtered to retain those with allele frequencies of < 1% in the Exome Variant Server, 1000 Genomes Samples, and Exome Aggregation Consortium database.¹² Variants were further filtered for de novo, recessive, and dominant inheritance patterns. The evidence supporting a diagnosis was then manually evaluated by comparison with the published literature. Analysis, interpretation, and reporting required an average of 6 h of expert effort. If rWGS or rWES established a provisional diagnosis for which a specific treatment was available to prevent morbidity or mortality, this was immediately conveyed to the clinical team. All causative variants were confirmed by Sanger sequencing or chromosomal microarray, as appropriate. Secondary findings were not reported, but medically actionable incidental findings were reported if families consented to receiving this information.