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(Miller et al., 2021; ACMG SF v3.1, unpublished ), The Clinical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.