Diagnostic whole exome sequencing of the proband only
|Turn-Around-Time (TAT)*||5 weeks|
|Acceptable Sample Types||
Whole Blood (EDTA)
Dried Blood Spots
|Acceptable Billing Types||
Self (patient) Payment
This test involves sequencing of the whole exome with a mean coverage of 100x, enhanced coverage of known disease-causing genes, and curated deep-intronic variants. Our WES test will reliably detect the majority of copy number variations (CNVs) of 3 exons or greater. Smaller CNV events may also be detected and reported, but additional follow-up testing is recommended if a smaller CNV is suspected. The WES assay will also detect microdeletion/duplication events greater than 500kb in clinically relevant regions, although follow-up testing may be warranted to better delineate the exact size of the event and confirm breakpoints. In addition to the primary analysis, patients can opt-in to a comprehensive secondary analysis including the recommended list by the American College of Medical Genetics and Genomics (ACMG).
- 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 an Agilent targeted sequence capture method to enrich the genes on this panel. Direct sequencing of the amplified captured regions was performed using 2X100bp reads on Illumina next-generation sequencing (NGS) systems. A base is considered to have sufficient coverage at 20X, and an exon is considered fully covered if all coding bases plus three nucleotides of flanking sequence on either side are covered at 20X or more. Low coverage regions, if any, are limited to ~1% or less of the nucleotides in the test unless a pathogenic variant explaining the phenotype is discovered. A list of these regions is available upon request. Alignment to the human reference genome (hg19) is performed, and annotated variants are identified in the targeted region. Variants are called at a minimum coverage of 8X and an alternate allele frequency of 20% or higher. Single nucleotide variants (SNVs) meeting internal quality assessment guidelines are confirmed by Sanger sequence analysis for records after results are reported. Indels and SNVs are confirmed by Sanger sequence analysis before reporting at the director's discretion. This assay cannot detect variants in regions of the exome that are not covered, such as deep intronic, promoter, and enhancer regions, areas containing large numbers of tandem repeats, and variants in mitochondrial DNA. Copy number variation (CNV) analysis is designed to detect deletions and duplications of three exons or more; in some instances, due to the size of the exons or other factors, not all CNVs may be analyzed. This assay is not designed to detect mosaicism; possible cases of mosaicism may be investigated at the discretion of the laboratory director. Primary data analysis is performed using Illumina DRAGEN Bio-IT Platform v.3.4.12. Secondary and tertiary data analysis is performed using PerkinElmer’s internal ODIN v.1.01 software for SNVs and Biodiscovery’s NxClinical v.6.1 or Illumina DRAGEN Bio-IT Platform v.3.4.12 for CNV and absence of heterozygosity (AOH). Genes and/or exons located in pseudogene regions are not covered in this assay. The StepOne Comprehensive Biochemical Profile is performed by tandem mass spectrometry as well as other technologies.
EDTA (purple top)
Infants (< 2-years): 2 to 3 mL; Children (>2-years): 3 to 5 mL; Older children and adults: Minimum 5mL. The blood tube should be inverted several times immediately after blood collection to prevent coagulation.
Clotted or hemolyzed samples are not accepted.
Required DNA Quantity by Test Type*:
- Next Generation Sequencing (NGS): Send >1000 ng total gDNA @ >15 ng/μL. Please ship samples in 10mM Tris. Do not use EDTA.
- Sanger Sequencing: Send >500 ng total gDNA @ >15 ng/μL (varies by the size of the gene and the variants requested).
- Non-Sanger Sequencing Tests: Send >500 ng total gDNA @ >15 ng/μL.
- Research Laboratories: DNA extracted in research laboratories is not acceptable. Only under exceptional circumstances (e.g., proband not available) will DNA extracted in a research laboratory be accepted for clinical testing. Additional testing (e.g., of other family members) may be required to confirm results.
- Laboratories outside the United States: Non-US laboratories are not subject to CLIA regulations and will be reviewed on a case-by-case basis. Please call to speak with a laboratory genetic counselor prior to submitting a DNA sample from any non-CLIA certified laboratory.
- Special Notes: If extracted DNA is submitted, information regarding the method used for extraction should be sent along with the sample.
Dried blood spot card
Follow kit instructions. Briefly, allow blood to saturate card until indicated areas are filled and blood has soaked through card. Air dry card at ambient temperature for at least 3 hours.
- NBS: Please contact PKIG to request the StepOne® kit.
- Gene Sequencing: Please contact PKIG to request the DBS collection kit.
- For pre-punched DBS: The required minimum 6 punches with 3.2 mm or 4 punches 4.75 mm.
Oragene™ Saliva Collection Kit or ORAcollect-Dx kit
Collect saliva on an Oragene™ Saliva Collection Kit ORAcollect-Dx kit according to the manufacturer’s instructions.
Please contact PerkinElmer to request the saliva collection kit for patients that cannot provide a blood sample as whole blood is the preferred sample.