Clinical ultra- rapid whole-genome sequencing (urWGS), rapid whole-genome sequencing (rWGS), and rapid whole-exome sequencing (rWES) were performed in laboratories accredited by the College of American Pathologists and certified through the Clinical Laboratory Improvement Amendments. 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 pre- pared 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 3 101 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. 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). 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).
Clinical ultra- rapid whole-genome sequencing (urWGS), rapid whole-genome sequencing (rWGS), and rapid whole-exome sequencing (rWES) were performed in laboratories accredited by the College of American Pathologists and certified through the Clinical Laboratory Improvement Amendments. 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 pre- pared 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 3 101 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. 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). 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).