Whole Exome Sequencing: Trio Analysis Print

WSLH Department: Cytogenetics
WSLH Test Code: 895M66
Availability: Monday-Friday 7:45 AM - 4:30 PM, Saturday 7:45 AM - 12:00 PM
Turn-around Time: 4-6 weeks
Recommended Uses: Establishing a diagnosis in individuals with a known or suspected genetic disorder for timely treatment and optimal outcomes; establishing a diagnosis in individuals with negative previous genetic testing (karyotype, chromosomal microarray analysis, single gene or gene panel testing).
Additional Tests Performed:
Patient Preparations:
Specimen Requirements: Blood:
Infants (0-1 year): 2 mL whole blood collected in EDTA (purple-top) tube(s).

Adults and Children: 5 mL (minimum) to 10 mL (preferred) whole blood collected in EDTA (purple-top) tube(s).

Saliva: Isohelix™ Saliva Collection kit used according to manufacturer instructions.

Buccal: Stabilized buccal swabs collected with SafeCollect kit (Zymo) according to manufacturer instructions.

Dried Blood Spot: At least one circle completely filled and dried on Whatman FTA™ cards.

DNA: 3-5 ug DNA in TE buffer at a concentration of 50-100 ng/uL. For best results, DNA should be treated with RNAse. DNA must be extracted in a CLIA-certified Laboratory OR a laboratory meeting equivalent requirements as determined by the CAP and/or the CMS.
Specimen Handling & Transport: Store and transport specimens at room temperature (may transport with coolant during hot weather, >85 degrees F). DO NOT FREEZE. The laboratory must receive specimens within 24-48 hours of collection.
Collection Kit/Container:
Collection Instructions: Blood: Draw blood using aseptic techniques into a sterile EDTA vacuum type tube(s). Invert tube(s) to mix. If using larger tubes, draw to full volume to avoid over-treatment with anticoagulant.

Saliva/Buccal: Collect according to manufacturer instructions.

DNA: DNA extracted from peripheral blood, cord blood, buccal swab, saliva, fresh and frozen tissue (postnatal), dried blood spots. We DO NOT accept DNA from Formalin-Fixed Paraffin-Embedded (FFPE) Tissue. Label with the patient name plus a second identifier. DNA concentration and volume must also be provided on the specimen label.
Unacceptable Conditions: Blood that is clotted or hemolyzed is not acceptable. Blood must not be frozen. Plasma and serum are not acceptable.
Requisition Form: Cytogenetics Lab Clinical Exome Sequencing Forms
Cytogenetics Lab Genetic Diagnosis Form #131
Required Information: Laboratory regulations require the following minimum information to be provided on the requisition form for a specimen to be accepted for testing: Patient name or unique identifier; date and time of collection, patient date of birth and sex, specimen type/site of collection, test request(s), reason for referral (detailed phenotype information), clinician name and UPIN/NPI, and address for reporting results. Please be certain that name/identifier on the form matches that on the specimen label. Clinical Exome Sequencing Forms: Medical and Family History Form, Consent form, Genetic Diagnosis Request Form
Results Include: Reports will include:

1. Exome primary findings: clinically relevant variants in genes that are associated with the diagnostic indication for which the exome was ordered.

2. Exome secondary findings: medically actionable diagnostic findings not related to the patient's phenotype (if requested). Secondary findings are limited to pathogenic or expected pathogenic variants identified in genes recommended by the ACMG (PMID: 23788249, PMID: 25356965).

3. Carrier status for pathogenic or likely pathogenic variants that are causative for recessive disease (if requested).
Limitations: This assay is designed to cover protein coding regions only and variants that lie within introns and untranslated regions may not be detected. At this time, whole exome sequencing cannot detect single and multi-exon deletions or duplications. In addition, large genomic rearrangements may not be detected unless breakpoints occur within the sequenced region. Trinucleotide repeat expansions and epigenetic changes such as DNA methylation are not evaluated. Whole exome sequencing does not provide complete coverage of all coding exons. Low-level mosaic variants (present in less than 25% of nucleated cells) may not be detected.
Additional Tests Recommended: Chromosomal microarray analysis (CMA) is recommended for the detection of copy number variations.
Additional Comments: See Cytogenetics website for forms
Methodology: Genomic DNA is isolated and the coding regions of targeted genes plus ~10 bases of non-coding DNA flanking each exon are captured and enriched using custom hybridization probes manufactured by IDT. Captured DNA is sequenced on the NovaSeq 6000 using 2x150 bp paired-end reads (Illumina, San Diego, CA, USA).

DNA sequences are aligned and compared to reference gene sequences based on human genome build GRCh37/hg19. A custom bioinformatics analysis pipeline is used to compare sequence changes (variants) to the reference sequence. Variants with a minimum coverage of 10X are analyzed.

Variant annotation and sorting is performed using BioDiscovery's NxClinical software. Each variant is evaluated using databases (e.g., ClinVar, gnomAD, ExAC, etc), published literature, clinical correlation, segregation analysis, and predicted functional or splicing impact (using computational tools such as PolyPhen, SIFT, etc). Variant classification follows ACMG guidelines (PMID:25741868). Interpretations may change over time as more information becomes available.

All reported sequence variants are confirmed by Sanger sequencing or an appropriate alternate method. Limits of detectable mosaicism for nuclear variants have not been determined. A portion of this test is run at Prevention Genetics (CLIA#52D2065132, CAP#7185561), 3800 S. Business Park Ave, Marshfield, Wisconsin 54449. A portion of this test is run in UWCS facility CLIA#52D2089533 425 Henry Mall, Madison, WI 53706.
Includes: Whole Exome Sequencing (WES) is used to detect variants in protein-coding DNA. The exome will be sequenced to an average read depth of >120x. Over 97% of the exome will be fully covered at ≥20X read depth.
CPT Code: 81415, 81416(x2)
Price: For pricing information, please call 608-262-0402.
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