Background:
The human exome encompasses all protein-coding regions of the genome, representing approximately 22,000 genes. It is estimated that about 85% of disease-causing mutations reside within these coding regions, making the exome a highly informative target for genetic analysis. Our clinical whole exome sequencing (WES) test is designed to provide comprehensive sequencing coverage across the entire exome, with enhanced coverage for genes known to be associated with disease. Exome sequencing offers significant clinical utility, particularly in diagnosing rare or complex hereditary disorders, resolving cases with inconclusive results from targeted panel testing, and evaluating individuals with multiple or atypical phenotypes. In addition, WES can uncover novel or previously unrecognized genetic conditions that may not be captured by more limited testing approaches. It can be ordered early in a proband’s (patient’s) evaluation, or following extensive negative genetic or metabolic tests.
Concurrent analysis of parents, or other comparators (e.g. affected siblings), by sequencing allows for improved interpretation through inheritance-based analysis, helping to clarify the clinical significance of variants that may underlie the patient’s condition.
Reasons for Referral:
- Resolving inconclusive results from targeted panel testing.
- Expediting diagnostic results.
- Evaluating individuals with a clinical presentation of multiple unexplained or atypical symptoms.
- Identifying a genetic etiology that may be contributing to a patient’s symptoms.
- Diagnosing rare or complex hereditary disorders.
Methodology:
Genomic DNA is analyzed using next-generation sequencing (NGS) on the Illumina NextSeq 2000 platform, with target enrichment performed using hybridization-based probes to capture exonic (coding) regions of the gene(s). Single nucleotide variants (SNVs) and small insertions or deletions (INDELs) are identified using the Illumina DRAGEN Enrichment Workflow, executed onboard the NextSeq2000. This pipeline combines software and hardware acceleration to generate high-confidence germline haplotype calls. Clinical and analytical validation of DRAGEN was performed in our laboratory. Based on validation study results, for SNVs, this assay achieves >96% analytical sensitivity and >99% positive predictive value (PPV). For INDELs <50 bp, the analytical sensitivity is >87% and the PPV is >97%. INDELs >50 bp may be detected but the sensitivity for these is reduced.
Exon-level copy number variants (CNVs) are detected using the Germline Copy Number Variation Best Practices pipeline from GATK. A Bayesian model, clinically validated in our laboratory, enables detection of deletions and duplications involving three or more contiguous exons in genes with adequate probe coverage and without complicating factors (e.g. pseudogene homology, short tandem repeats, segmental duplications). Please note that exon-centric microarray remains the gold standard for exonic copy number variant calling. If exon-centric microarray is of interest, please contact the laboratory for additional information.
This test is not designed to detect polynucleotide repeats, low-level mosaicism, structural rearrangements or balanced alterations (e.g. inversions, gene conversion events, translocations, etc.) or variants in difficult regions. Additionally, variants located in regions of insufficient coverage, including introns and promoter regions; pseudogenes; where the reference genome is inaccurate or contains gaps and insertions; and of high GC content may not be detected. This test does not provide complete coverage of all exons and noncoding regions may have limited information and ability to interpret. Variants in introns that are greater than 10 bp from the intron-exon junction may be analyzed. Please contact the laboratory if interrogation of intronic sequence greater than 10 bp from the intron-exon boundary is desired.
Specimen Requirements:
Three (3) samples to be collected:
- Proband
- Biological Relative #1
- Biological Relative #2
Blood: EDTA or ACD (Solution A or B):
- Adult: 5mL
- Child: 5mL
- Infant: 2-3mL
Saliva: 2 ORAgene Saliva Kits (OGR-500) used according to manufacturer instructions. Please contact KDL Client Services for a Saliva Collection Kit for patients that cannot provide a blood sample.
Assisted Saliva: 4 ORAgene Assisted Saliva Kits (OGR-575) used according to manufacturer instructions. Please contact KDL Client Services for an Assisted Saliva Collection Kit for patients that cannot provide a blood sample.
Buccal Cells: 4 CytoSoft™ Cytology Brush (Medical Packaging CYB-1) used according to manufacturer instructions. Please contact KDL Client Services for a Buccal Collection Kit for patients that cannot provide a blood sample.
- Please note that buccal samples may yield a limited amount of DNA, and additional specimens may be required for confirmatory or supplementary testing.
DNA: 10µg at a minimum of 60-100ng/µL (DNA must be extracted in a CLIA-certified laboratory or a laboratory meeting equivalent requirements as determined by the CAP and/or CMS)
For routine testing of blood and saliva (or DNA extracted from them), KDL does NOT accept samples from patients within two (2) weeks of a packed cell/platelet transfusion or within four (4) weeks of a whole blood transfusion. For extraordinary circumstances, where testing must be performed within the above windows, please contact our lab.
A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES. Please include detailed clinical information, including ethnicity, clinical history, and family history.
Exome Sequencing Consent Form must be filled out and must accompany all samples.
Test Performed (Days):
Weekly
Turn Around Time:
Approximately 8-10 weeks
Shipment Sensitivity Requirements:
- Package and ship specimen to remain cold, but not frozen.
- Ship via overnight express, using the FedEx priority overnight label provided.
- Contact Client Services for shipping kits and instructions at (855) 535-1522.
References:
Additional Info: