Genome Analysis and Interpretation
GS was performed using next generation sequencing on the Illumina NovaSeq 6000 platform through The Center for Applied Genomics (TCAG) at the Hospital for Sick Children in Toronto, Ontario. Data processing and analysis was performed using two different pipelines: the Franklin Genoox platform (genoox.com; GRCh37/hg19 alignment) and locally following Genome Analysis Toolkit best practices (GATK 3.7; GRCh38/hg38 alignment). Filtration parameters were developed to capture substitutions, small or large insertions, deletions, duplications or indels 1) previously classified as likely pathogenic or pathogenic in ClinVar®; 2) with aggregated and/or internal allele frequencies ≤5%; and, 3) in genes with established gene-disease relationships. Copy number variants (CNVs) were filtered for inclusion based on 1) OMIM morbid genes; 2) ISCA triplo- or haplo-sensitivity scores; 3) size (>20Kb); and, 4) frequency (<1%). CNVs with >90% overlap with known benign, polymorphic CNV regions were excluded. Variants were further filtered by quality parameters such as confidence of calls, availability of published evidence, and participant preferences for SF.
Comprehensive gene panels were developed to aid in variant filtration and assessment. The medically actionable gene panel was developed according to the ACMG list of reportable SF versions 3.0 and v3.1 (Miller et al., 2021; ACMG SF v3.1, unpublished ), The Clinical Genome Resource (ClinGen) (Rehm et al., 2015), and Reble et al. (2021), for a total of 208 genes. Reporting of medically actionable variants was not limited to the genes in this list and was subject to inclusion upon review by the study team. All other genes (n=9965) associated with carrier status and rare Mendelian conditions were included in comprehensive disease panels developed through referencing publicly available gene data sets (Table 1), including ClinGen (Rehm et al., 2015), the Clinical Genomics Database (Solomon et al., 2013), Online Mendelian Inheritance in Man® (OMIM), BabySeq (Ceyhan-Birsoy et al., 2017), and The Gene Curation Coalition (GenCC) (DiStefano et al., 2022, Preprint. DOI: https://doi.org/10.1101/2022.01.03.21268593) gene databases. For the complete gene database please refer to Supplementary File 1.
The study team followed a standard operating procedure for novel variant assessment, which included stepwise processes for applying pre-defined filters and accumulating evidence for or against pathogenicity based on ACMG guidelines (Richards et al., 2015). Variants determined to be benign, likely benign, or of uncertain significance were excluded from the final report. CNVs were reported in the context of personal disease risks only. Publicly available sources including GeneReviews® (Adam et al., 1993-2022) and OMIM® (https://www.omim.org/) were used to compile disease and familial risk information.