Reflex to Whole Genome Sequencing (from genome panels)

• Genetically heterogeneous disease caused by likely pathogenic/pathogenic findings in multiple genes
• Condition suggestive of a genetic disorder with a long differential diagnosis list
• Unclear or atypical presentation of a genetic disorder
• Previous genetic testing did not yield a diagnosis

Sequencing is performed on genomic DNA using a targeted sequence capture method to enrich for the exome. Captured regions are sequenced on a short-read next-generation sequencing (NGS) system. Coverage requirements are based on validation data. An exon is considered fully covered if all coding bases plus three nucleotides of flanking sequence on either side are covered. Alignment to the human reference genome is performed, and annotated variants within the targeted region are identified. Variants are reviewed based on minimum coverage and alternate allele frequency cutoffs defined as per laboratory procedure. Indel and single-nucleotide variants (SNVs) may be confirmed by Sanger sequencing analysis before reporting, based on laboratory requirements.

This assay cannot detect variants in regions that are not targeted, including deep intronic, promoter, or enhancer regions. Exons with nonunique sequences, such as those containing pseudogene regions, are not analyzed in this assay. Copy number variation (CNV) analysis detects deletions and duplications in genes on this panel; however, in some instances, due to exon size, sequence complexity, or other factors, certain CNVs may be difficult to detect and analyze. When reported, copy number variant size is approximate, and actual breakpoint locations may lie outside of the targeted regions. Only CNVs identified in genes included in the panel will be reported. This assay does not interrogate CNVs in mitochondrial DNA. CNV analysis will not detect tandem repeats, balanced alterations (reciprocal translocations, Robertsonian translocations, inversions, and balanced insertions), methylation abnormalities, triploidy, or genomic imbalances in segmentally duplicated regions. This assay is not designed to detect mosaicism; however, possible cases of mosaicism may be detected and reported if laboratory requirements are met.

Primary data analysis is performed using standard FASTQ conversion tools appropriate to the sequencing platform. Secondary analysis is conducted using a high-performance genomic analysis pipeline. Tertiary analysis incorporates established annotation tools together with Revvity Omics' internal software. Copy number variation and absence of heterozygosity assessments are performed using a clinical cytogenomics platform.