What is exome sequencing and when is it useful?
Exome sequencing targets the protein-coding regions, called exons, of the approximately 20,000 genes in the genome. It is a powerful diagnostic tool, providing a definitive diagnosis in 20-50% of patients (Yang, et al. N Engl J Med. 2013 Oct 17;369(16):1502-11, and GeneDx, Retterer et al., Genet Med. 2015 Dec 3). Exome sequencing can be used to identify the molecular basis of a genetic disorder in individuals:
- With a genetically heterogeneous disease as pathogenic findings could be present in many different genes
- With a long list of differential diagnoses
- With an atypical presentation of a genetic disorder
- Who have exhausted other currently available genetic testing options
- Who have suspected tissue-specific mosaicism
What does GeneDx need to perform XomeDx® testing?
- Proband’s specimen. Please visit the Specimen Requirement Page for more information.
- Family member samples, if appropriate depending on Xome test requested.
- Completed XomeDx® Test Requisition Form and signed Informed Consent Form.
- Medical records, including prior test results, consult notes, and pedigree/family history. Client is encouraged to provide specific genes/disorders of interest and differential diagnosis.
What is the cost of XomeDx®?
Questions regarding pricing and billing can be directed to GeneDx Customer Service at zebras@genedx.com or by calling 1-888-729-1206.
How long will it take to receive XomeDx® results?
The turnaround time will vary depending on the specific Xome test requested. Please see the specific test page for additional information.
Whose specimens should be sent for XomeDx® and what testing is performed?
When a trio (proband plus both parents) is sent for XomeDx® and XomeDx®Plus, exome sequencing is always performed on each member of the trio. This improves the sensitivity of the analysis when compared to testing only the proband. In some cases, including siblings or other relatives of the proband can be helpful. GeneDx will decide, in consultation with the ordering provider, which individuals will optimize our ability to identify a genetic cause of the patient’s features. We will also discuss the type of testing that is most useful and appropriate (i.e. exome sequencing or targeted variant testing to determine segregation). All individuals’ samples should be submitted at the beginning of testing. Relative samples may be collected and shipped separately from the proband sample; however, relative specimens must be received within three weeks of receipt of the proband's sample. In general, only a single report will be issued for the proband. However, the report will describe the inheritance and segregation of variants for all tested individuals.
Although exome sequence data may be generated on relatives, this data is only used to aid in the analysis of the proband's data. GeneDx does not conduct an independent analysis on relative samples. A second, independent analysis on a relative can be ordered, additional fees apply.
Can I send a different relative if a parent is unavailable?
Biological parents are typically the most informative samples and are accepted whenever possible. If parents are unavailable, other relatives may be considered on a case by case basis. To review details of a specific case, you can contact a GeneDx Clinical Genomics Genetic Counselor at zebras@genedx.com or 1-888-729-1206.
Is exome sequencing via a trio better than a proband alone?
The positive rate is highest when a trio is submitted/analyzed, as the inclusion of parental data improves the ability to classify variants. The absence of parental data may lead to over reporting of variants of unknown significance that could otherwise be dismissed based on inheritance patterns.
Can you use XomeDx® to screen the parents for carrier status of recessive diseases?
Carrier testing for autosomal recessive conditions is best performed via other tests, such as ReproXpanded and/or GenPath: Inherigen.
How does GeneDx identify variants associated with the patient’s phenotype?
Exome sequencing will identify hundreds of thousands of variants. Variants are filtered using a variety of factors including population frequency, presence of gene and/or variant in HGMD or other databases, inheritance pattern, phenotype, severity of sequence change, and function in pathways. The clinical information provided by the ordering provider, including a description of the features, family history, and prior test results, is critical in the analysis of variants.
What will the test report include?
A single XomeDx® report will be issued on the proband/main affected individual in the family. A separate report will not be issued for family members who may also have submitted a specimen for the purpose of allowing better interpretation of the results from the affected individual. If additional reports are requested for other affected family members, additional fees will apply. The XomeDx® report issued for the affected individual will contain variations in genes previously implicated in a human disease similar to the affected individual or in genes hypothesized to be related to the cause of the disease (candidate genes), based upon the function, tissue of expression, and phenotype of model organisms with alterations in the gene. Variants in candidate genes may also be reported based on internal data, such as observations of previous XomeDx® cases with similar phenotypes and types of variations in the same gene.
What are ACMG Secondary Findings?
The American College of Medical Genetics and Genomics (ACMG) recommends that secondary findings identified in a specific subset of genes associated with medically actionable, inherited disorders be reported for all probands undergoing exome sequencing, specifically when these variants are known and/or expected to be disease-causing.
Please refer to the latest version of the ACMG Recommendations for Reporting of Secondary Findings in Clinical Exome and Genome Sequencing Report for complete details of the genes and associated genetic disorders. If appropriate for the specific XomeDx test, secondary findings will be included in the test report, unless a proband opts-out of receiving this information in the Patient Consent portion of the XomeDx® Test Requisition Form. The presence of any secondary finding(s) reported for the affected individual will be provided for all relatives tested by XomeDx® or XomeDx®Plus. GeneDx does not conduct an independent evaluation of secondary findings in relatives as part of the proband's XomeDx® test. Relatives have the ability to opt-out of receiving secondary findings. Secondary findings will be confirmed by an alternate test method.
Can I request a separate report for family members that includes ACMG secondary findings not present in the proband?
Yes, as a separate test. An independent analysis for ACMG secondary findings can be ordered via XomeDxSlice, test code 706. ACMG secondary findings are also reported as part of XomeDx®Insights, an exome based test designed for generally healthy adults who would like to learn about medically relevant changes in their genetic code, and their risk to have or develop certain genetic conditions.
Are there any findings that will NOT be reported?
A single report will be issued on the main affected individual in the family, referred to as the proband. No separate reports will be issued for the parents or other unaffected relatives. Variants known to be benign (not associated with any disease) and/or commonly seen in many other healthy people will not be reported. If a patient opts-out of receiving secondary findings, we will not analyze or report variants in the ACMG-recommended genes unless they are related to the patient’s phenotype. Only variants associated with the proband's reported clinical features will be reported and/or ACMG secondary findings if the family opted-in.
Will the analysis identify variants in disease-associated genes that are not associated with the patient’s reported phenotype?
Given the large number of genes analyzed via exome sequencing, pathogenic variants may be detected in genes that may be medically significant, but not associated with the primary reason for testing in a given patient.
In rare cases, GeneDx may report an incidental finding in a gene that is not one of the genes recommended by the ACMG. These reported findings must be pathogenic variants identified in the coding exons of genes, considered medically actionable, with the results expected to have a significant impact on the patient's health. These findings are not reported without prior discussion with the ordering clinician.
Are there genes that are not well covered by this method?
It is possible that entire genes may not be able to captured and sequenced in a particular patient, though in general we expect that there are only small portions of different genes not amenable to evaluation. Some exons have low or no coverage because no probes have been designed or are unavailable for these regions. It may be challenging to obtain usable sequence data for some regions. To check coverage of specific genes via exome sequencing, visit the XomeDx®Slice Tool.
What sequencing platform and capture kit is GeneDx using?
GeneDx uses an Illumina sequencing platform and IDT xGen v1.0 capture.
What is the methodology?
Genomic DNA from the submitted specimen is enriched for the complete coding regions and splice site junctions for most genes of the human genome using a proprietary capture system developed by GeneDx for next-generation sequencing with CNV calling (NGS-CNV). The enriched targets are simultaneously sequenced with paired-end reads on an Illumina platform. Bi-directional sequence reads are assembled and aligned to reference sequences based on NCBI RefSeq transcripts and human genome build GRCh37/UCSC hg19. Using a custom developed analysis tool (XomeAnalyzer), data are filtered and analyzed to identify sequence variants and most deletions and duplications involving three or more coding exons (Retterer et al., 2015). Smaller deletions or duplications may not be reliably identified. Reported clinically significant variants are confirmed by an appropriate orthogonal method in the proband and, if submitted, in selected relatives as necessary. Reportable variants include pathogenic variants, likely pathogenic variants and variants of uncertain significance. Likely benign and benign variants, if present, are not routinely reported.
Can I get the capture target .bed file?
GeneDx exome sequencing uses IDT xGen v1.0 and the bed file for the probe and target locations are available directly from IDT: https://www.idtdna.com/pages/products/next-generation-sequencing/hybridization-capture/lockdown-panels/xgen-exome-research-panel (near the bottom of the page under Resources).
What are the statistics for coverage/quality?
Approximately 98% of the targeted region of an affected individual's exome will be assessed with the XomeDx test at a minimum of 10x coverage, the minimum read depth necessary to detect a variant. Across the exome, the average depth of coverage is 100-120x. The test report will include case-specific exome coverage. Average read depth statistics for the XomeDx test are as follows:
| Read Depth |
10x |
20x |
30x |
40x |
50x |
| Mean Percent of Target Covered |
98% |
97% |
94% |
90% |
83% |
Does exome sequencing detect copy number variants (i.e. deletions, duplications)?
Since 2014, GeneDx has been detecting copy number variants (CNVs) directly from exome sequencing data using an in-house developed algorithm (Retterer et al., 2015, PMID 25356966). This method can reliably detect most deletions and duplications involving three or more coding exons; smaller deletions or duplications may not be reliably identified. All reported CNVs are confirmed by an orthogonal method such as array CGH, MLPA, or PCR and parental confirmations for reportable CNVs are reported without additional charges. CNV analysis may not be possible in approximately 5% of samples. All Xome-based reports will include whether CNV detection was possible. At this time, exome based technology is not a replacement for the sensitivity of exon level aCGH.
Our CNV analysis via XomeDx® can identify:
- Large multi-gene chromosomal aberrations
- Small, partial gene, and intragenic CNVs; deletions and duplications of three exons or larger are reliably identified
- CNVs of one to two exons in size may be identified and comprise >50% of our previously reported exon-level variants
- Chromosomal aneuploidy
- Uniparental disomy, including isodisomy and heterodisomy
- Inheritance of CNVs when a trio is submitted
CNV analysis is limited or may not be possible in regions with reduced coverage, extreme GC- or AT-content, or significant homology to pseudogenes or segmental duplications. Our method may not always detect: balanced aberrations, mosaicism, deletions/duplications involving only part of an exon, or CNVs in non-coding regions of the genome.
My patient's test report was negative but I know a genetic condition is present in the family. What additional testing can be requested?
A negative report means we did not detect any variants related to the reported clinical features in any area of the exome that was tested at the time of the analysis. It certainly is possible that the cause of the affected individual’s disease is due to an underlying genetic condition. There could be a variant in a region of the exome that is not covered by this test, a type of variant that cannot be detected by the exome test, or there may be a variant that is observed in a gene not yet known to cause human disease. Depending on the specific situation and condition of interest in a given family, additional genetic testing may be warranted.
Reanalysis of the previously generated exome data can be considered, since technology and bioinformatics pipelines continuously improve and new disease genes are published.
What is reanalysis and when should I request this?
Reanalysis is the process of reexamining the sequencing data generated from the XomeDx® test. This is most commonly requested when the initial exome sequencing did not yield a definitive result and may be especially helpful in cases where there are changes to the phenotype. Generally, reanalysis is recommended to occur at least one year after the date of the initial XomeDx® report. Currently, we offer a one-time, no-charge reanalysis for every XomeDx® or XomeDx®Plus test.
What if extended family members want to be tested for a variant which was identified in a patient by XomeDx® testing?
Family members can be tested for variants identified in the initial patient. If the variants detected in the proband are classified as pathogenic or likely pathogenic, family member testing can be ordered via Targeted Variant Testing (Site-Specific).
Laboratories classify genetic changes as variants of uncertain significance (VUS) if there is incomplete or conflicting information about the health consequences of the variant. In some cases, testing family members for the presence or absence of the VUS may contribute to a better understanding of the variant and may be one piece of evidence leading to eventual reclassification of a VUS as a pathogenic, likely pathogenic, benign, or likely benign variant. For such cases, GeneDx has established a Variant Testing Program (VTP). GeneDx considers requests for the Clinical Genomics VTP for any individual found to have a VUS in a disease-causing gene through exome or genome sequencing at our laboratory. These studies will be performed at no additional charge for select and pre-approved family members who meet certain criteria and for whom appropriate clinical information is provided. Genetic testing for other variants or additional family members, including predictive testing, is not included in our VTP and can be ordered separately for charge. GeneDx will make the final determinations for VTP in its sole discretion.
Inquiries regarding the Clinical Genomics VTP can be directed to GeneDx Clinical Genomics genetic counselors via email zebras@genedx.com, phone 301-519-2100, or fax 301-519-2892, that includes a detailed pedigree and any relevant clinical information/evaluations. Please be sure to indicate that you are submitting the information for VTP consideration, and include the name and/or GeneDx accession number of the proband.
I still have questions regarding an XomeDx® test. Who should I contact?
You can contact a Clinical Genomics Genetic Counselor through our Customer Service team:
