Diagnostic Exome Sequencing Test Details
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Background
The Diagnostic Exome Sequencing test aims to end the diagnostic odyssey for individuals with rare genetic disorders, ultimately guiding clinical care for patients and their families. This test provides physicians with high quality exome sequencing, as well as unparalleled data interpretation. Results will be returned to the ordering physician in a concise report featuring an overall test result and in-depth phenotype-driven interpretation of known or plausible genetic causes of disease. Patients have the option to expand the report to include clinically actionable secondary findings (unrelated to the primary indication for testing) in 59 genes.
Diagnostic Exome Sequencing Consultation
If you would like assistance in determining the appropriateness of diagnostic exome sequencing for your patient, you may contact us by phone or submit pages 2–3 of our requisition form, along with your contact information, and we will contact you.
Indication for Testing
Testing is currently recommended for individuals with a suspected genetic disorder in whom traditional genetic testing has not yielded a result, despite a suspected genetic etiology. Exome sequencing may also be considered as a first-line testing strategy for conditions with a high degree of genetic heterogeneity for which panel-based testing is either limited or unavailable.
Methodology
This assay is performed using the PerkinElmer Sciclone® G3 Workstation combined with the Agilent SureSelect Clinical Research Exome capture kit (#G9496A 5190-7344) followed by sequencing of the coding regions and splice sites on the Illumina NextSeq 550 (High-Output v2 kit). Exomes are sequenced to achieve a completeness > 95% of bases covered with at least 15 reads across the entire exome. Reads are aligned to the GRCh37 reference sequence using the Burrows-Wheeler Aligner (BWA 0.7.17) and variant calls are made using the Genomic Analysis Tool Kit (GATK v4.0.3.0). Variants are subsequently filtered to identify: (1) variants classified as disease causing mutations in public databases with minor allele frequencies <5.0% in the Genome Aggregation Database (gnomAD, http://gnomad.broadinstitute.org/); (2) nonsense, frameshift, and canonical splice-site variants in disease-associated genes with a minor allele frequencies ≤1.0% in gnomAD; and (3) variants with minor allele frequencies ≤5.0% in gnomAD in a patient-specific phenotype-driven gene list. The evidence for phenotype-causality is then evaluated for each variant resulting from the filtering strategies above and variants are classified based on ACMG/AMP criteria (Richards et al. 2015) with ClinGen rule specifications (https://www.clinicalgenome.org/working-groups/sequence-variant-interpretation/). Variants are reported according to HGVS nomenclature (https://varnomen.hgvs.org/). Only variants with evidence for causing or contributing to disease or variants of uncertain significance in genes highly related to the reported patient phenotype are included on the final report. All variants included on this report are confirmed via Sanger sequencing or another orthogonal technology.Analytical and Clinical Sensitivity
This test is 98.88% sensitive (95% CI =98.84-98.91%) to detect variants changing a single base and 92.31% sensitive to detect insertion/deletions (95% CI =91.68-92.94%) across the entire exome. Technical positive predictive value for single nucleotide variant changes is 99.37% (95% CI = 99.31-99.43%) and 93.94% (95% CI = 93.09-94.80%) for insertion/deletion changes. There is demonstrated reduced detection for larger indels, especially in low complexity regions with corresponding low sequence coverage, and for variants in regions with high homology.Limitations
Specific types of genetic variation, such as repeat expansions, translocations, inversions, copy number events, and mitochondrial variation are currently not reliably detected by exome sequencing and are, therefore, not included in this assay. Because diagnostic exome sequencing only covers the coding regions of the genome, any genetic changes residing outside of the targeted region will not be detected. Furthermore, not all coding regions are sufficiently covered and variants in regions of low coverage may not be reliably detected. Gene-level coverage information for both tests is available online and details regarding the coverage of genes associated with a specific indication can be determined upon request.
For additional questions, you also may visit our Frequently Asked Questions page.
Interested in Exome or Genome Sequencing
For further information, please contact the
Laboratory for Molecular Medicine
Phone: 617-768-8500
Fax: (617) 768-8513
Email: lmm@partners.org
Amy Hernandez, MS, CGC
Director of Genetic Counseling
Phone: (617) 768-8516
Email: alhernandez@partners.org
