Whole Exome Sequencing (WES) Trio

Whole Exome Sequencing (WES) Trio uses next-generation sequencing (NGS) to analyze all exons associated with rare and complex genetic disorders in the genetic material of two related individuals. It is a comprehensive genetic test specifically designed to support accurate diagnosis, risk assessment, and prevention.

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Whole Exome Sequencing (WES) Trio is presented as a comprehensive, hypothesis-free genetic test designed for the simultaneous analysis of three related individuals, typically the patient and both biological parents. The protein-coding regions of approximately 20,000 genes in the nuclear genome are analyzed, along with mitochondrial DNA, providing extensive genomic coverage. This approach enables the detection of multiple variant types, including single-nucleotide variants, small insertions and deletions (indels), as well as larger deletions and duplications. With this broad analytical scope, Whole Exome Sequencing (WES) is a powerful tool for identifying genetic alterations associated with diverse clinical phenotypes.

The exome is the portion of the genome that encodes proteins, which are essential for normal cellular structure and function. Although it comprises only about 1% of the entire genome, it contains approximately 85% of known disease-causing variants. This concentration of clinically relevant information makes the exome a critical target for genetic analysis. Variations within these regions can disrupt protein function, leading to a wide range of genetic disorders, thereby underscoring the importance of exome-focused sequencing in clinical diagnostics.

Whole Exome Sequencing (WES) is particularly valuable in cases where the clinical presentation is complex or atypical and multiple differential diagnoses are considered. It is frequently applied in the investigation of genetically heterogeneous conditions, where mutations in different genes may produce similar clinical features. The test is also indicated when previous genetic testing has yielded inconclusive results or when no specific diagnostic direction can be established. In such scenarios, Whole Exome Sequencing (WES) provides a comprehensive approach that extends beyond the limitations of targeted testing strategies.

A broad spectrum of genetic variation can be assessed through Whole Exome Sequencing (WES). This includes the identification of single-nucleotide variants, small insertions and deletions, and copy number variants such as deletions and duplications. In addition, variants within mitochondrial DNA are evaluated, enabling the detection of alterations affecting cellular energy metabolism. By simultaneously analyzing multiple types of genetic variation across thousands of genes, Whole Exome Sequencing (WES) offers a unified approach to exploring diverse molecular mechanisms underlying disease.

The clinical utility of Whole Exome Sequencing (WES) is reflected in its ability to increase diagnostic yield compared to more targeted genetic tests. The inclusion of parental data enables more confident interpretation of variant significance and supports informed clinical decision-making. Rare or previously unreported variants may be identified, contributing to a more precise understanding of disease etiology. This expanded diagnostic capability supports informed clinical decision-making and may guide personalized patient management over time. Furthermore, the information obtained can enhance the understanding of complex genetic conditions, facilitating more accurate prognosis and long-term care strategies.

Despite its comprehensive nature, certain limitations are recognized. Not all genetic variants can be reliably detected using Whole Exome Sequencing (WES), particularly those located outside coding regions or involving complex structural changes. The interpretation of identified variants may be challenging and is subject to ongoing refinement as scientific knowledge evolves. Results must therefore be considered in conjunction with detailed clinical information. It is also explicitly noted that this test is not intended for use in prenatal diagnostics.

Data generated through Whole Exome Sequencing (WES) are analyzed using advanced bioinformatics methodologies, ensuring high-quality and consistent interpretation. Variant classification is performed in accordance with current scientific evidence and internationally recognized standards, including established guidelines such as those from the American College of Medical Genetics and Genomics (ACMG). Continuous integration of emerging data from scientific literature and genomic databases supports the ongoing refinement of variant interpretation, ensuring that findings remain clinically relevant and up to date.

The test is performed in a clinical laboratory accredited to ISO 15189 and certified by CLIA and CAP, ensuring the validity, accuracy and international recognition of the results.

Genetic testing via Whole Exome Sequencing (WES) is performed by taking a blood sample, which can be done:

• At Diagnostiki Athinon - By taking a blood sample in Diagnostiki Athinon. There is no need for prior fasting. Book your appointment in real-time and purchase the test online

Test Strengths

The test is characterized by the following strengths:

• Analysis is performed in a CAP-accredited laboratory with the participation of CLIA-certified personnel.
• Advanced sequencing technologies, high-performance target enrichment methods, and precise bioinformatics pipelines ensure superior analytical performance.
• The assay is designed to provide comprehensive coverage of coding regions and clinically relevant splice-site regions.
• A rigorous variant classification system is applied.
• A systematic interpretation workflow, supported by proprietary software, enables accurate and traceable processing of NGS data.
• Comprehensive and clinically meaningful reports are provided.

Test Limitations

Specific technical and biological limitations apply:

• This test is not available for prenatal samples.
• Specific might be excluded from the analysis due to insufficient coverage by high-quality sequencing reads.
• Genes with suboptimal coverage are marked with a number sign (#), while those with partial or complete segmental duplications overlapping UCSC pseudogene regions are marked with an asterisk (*).
• A gene is considered to have suboptimal coverage when more than 90% of its target nucleotides are not covered at a depth of greater than 20x with mapping quality score (MQ > 20) reads. In such cases, detection of variants may be limited.

This test does not detect:

• Complex inversions
• Gene conversions
• Balanced translocations
• Repeat expansion disorders, unless otherwise specified
• Non-coding variants beyond ±20 base pairs from the exon–intron boundary, unless otherwise indicated
• The complete mitochondrial genome in all panels (see Panel Content section

This test may not reliably detect:

• Low-level mosaicism in nuclear genes (variants with a minor allele fraction of ~14.6% are detected with 90% probability)
• Stretches of mononucleotide repeats
• Insertions or deletions larger than 50 bp
• Small deletions or duplications, including single-exon events
• Variants within pseudogene regions or duplicated genomic segments

For further details, please refer to the Bioinformatics & Performance section.

Bioinformatics

The target region encompasses all protein-coding exons across the genome, including ±20 base pairs from exon–intron boundaries. When the test includes the mitochondrial genome, the target region also comprises the complete set of mitochondrial genes.

Sequencing data is processed through a proprietary analysis and annotation pipeline that integrates state-of-the-art algorithms and industry-standard software solutions. Multiple quality control steps are embedded throughout the workflow to ensure the accuracy, validity, and consistency of the results. The pipeline is optimized to maximize sensitivity without compromising specificity.

For variant interpretation, the system incorporates data from multiple reference populations and mutation databases, including (but not limited to) the 1000 Genomes Project, gnomAD, ClinVar, and HGMD. In silico tools such as SIFT, PolyPhen, and MutationTaster are also applied to support the classification of missense variants. Analysis is primarily focused on genes known to be associated with human disease, with continuous updates based on emerging scientific literature and curated databases. Splicing impact may also be evaluated using established computational tools.

All findings are summarized in a clear and detailed clinical report. This report includes sequencing quality metrics, gene-level coverage information, and highlights any regions where coverage is limited (<20x for nuclear genes and <1000x for mtDNA, when applicable). This ensures transparency and supports accurate clinical decision-making. Where applicable, additional analysis details and sequencing metrics may be made available through dedicated reporting platforms.

Test Performace

Whole exome sequencing enables the analysis of all protein-coding genes, with clinical interpretation primarily focused on genes supported by current scientific and clinical evidence.

The assay is based on a high-quality, clinical-grade whole exome sequencing (WES) approach.The assay is designed to achieve high and uniform sequencing coverage across clinically relevant regions of the exome. Details regarding the detection of different types of genetic alterations are provided in the sequencing and detection performance table (see Table).

The assay has been validated for several sample types, including EDTA blood, isolated DNA (excluding DNA from formalin-fixed paraffin-embedded tissue), saliva, and dried blood spots (filter cards). These sample types were chosen to maximize the probability of obtaining high-quality DNA. The diagnostic yield may vary depending on the assay performed, the referring healthcare professional, the hospital, and the country in which the test is conducted. The analysis includes detection of large deletions and duplications (copy number variants, CNVs), complementing sequence variant detection.

The performance metrics shown below are based on initial validation studies at an accredited clinical laboratory, with equivalent results confirmed across additional laboratory sites. Performance metrics are based on analytical validation studies and may not fully reflect performance in all clinical settings.

Performance of a high-quality, clinical-grade whole exome sequencing assay

Coverage metrics

Performance of Mitochondrial Sequencing Assay

Mitochondrial assay coverage metrics

Comprehensive clinical reports are delivered, designed to provide healthcare professionals with meaningful and actionable insights. Clinical interpretation requires a solid understanding of both clinical genetics and genetic principles. Reports are prepared by teams of molecular geneticists, medical experts, and other highly experienced professionals, who evaluate identified variants in the context of the patient’s phenotypic information provided in the requisition form.

For whole exome sequencing (WES), analysis is performed using a genotype-first (variant-driven) approach. Rather than restricting analysis to predefined gene panels, all protein-coding genes are evaluated, complemented by selected non-coding genes with established disease associations. This approach enables the identification of variants in genes not previously suspected based on the clinical presentation.

Analysis initially focuses on genes with well-established associations with human disease. If no conclusive findings are identified, further analysis includes candidate variants in genes not yet definitively associated with disease but with features suggestive of pathogenicity (e.g., de novo occurrence, predicted loss-of-function variants in constrained genes, or rare biallelic variants)

The goal is to ensure that reports are clinically valuable and understandable to all healthcare providers, regardless of their level of training in genetics and genetics-related fields. Variant classification forms the foundation of clinical interpretation and guides patient management decisions. Classifications follow the ACMG 2015 guidelines. Sequence and copy number variants classified as pathogenic, likely pathogenic, or of uncertain significance (VUS) are confirmed using bidirectional Sanger sequencing or orthogonal methods such as qPCR/ddPCR whenever NGS quality metrics do not meet strict thresholds for a true positive call.

Variant prioritization takes into account inheritance patterns consistent with the patient’s phenotype, including heterozygous variants in autosomal dominant genes, homozygous or compound heterozygous variants in autosomal recessive genes, and hemizygous or heterozygous variants in X-linked genes.

Each report includes tables for sequence and copy number variants, presenting essential information such as genomic coordinates, HGVS nomenclature, zygosity, allele frequencies, in silico predictions, phenotypic correlations, and variant classification. In addition, detailed descriptions are provided for the variant, the gene, and the related phenotypes, covering the gene’s role in human disease, mutation spectrum, variation in population cohorts, and links to associated conditions. References, abstracts, and variant databases are also cited, enabling healthcare providers to further evaluate reported findings if desired.

In the context of WES, reports may also include a separate section for secondary findings, where applicable (see below).

The primary findings consist of variants known to cause disease or rare variants that may explain the patient’s phenotype. The conclusion summarizes all available evidence and presents the rationale for classification.

Additional findings may include variants with limited or uncertain relevance to the clinical presentation. Examples include heterozygous variants in autosomal recessive genes, VUS in autosomal dominant genes (when a pathogenic or likely pathogenic variant already explains the phenotype), or risk alleles in genes included in the analysis.

Carrier status for variants unrelated to the patient’s phenotype is generally not assessed or reported

Secondary findings refer to pathogenic or likely pathogenic variants identified in genes unrelated to the primary indication for testing but associated with medically actionable conditions. These findings are reported in accordance with ACMG recommendations and only if the patient (or caregiver) has provided prior consent (opt-in). Secondary findings are reported in a separate section and, when applicable, separately for each tested family member.

The identification of pathogenic or likely pathogenic variants in dominant disorders, or biallelic variants in recessive disorders, is considered molecular confirmation of the clinical diagnosis. In such cases, family testing may assist in risk assessment. Variants of uncertain significance are not recommended for patient management or family risk stratification. Genetic counseling is strongly advised.

Accurate and detailed clinical information (including phenotype, age of onset, and family history) is essential for optimal interpretation of WES data and improves the likelihood of identifying clinically relevant variants.

Interpretation teams routinely analyze millions of variants across thousands of cases of rare diseases. With each case, internal knowledge of variant–phenotype relationships is enriched, enabling continuous refinement of classifications. As new evidence becomes available, previously reported variants may be reclassified. If a variant initially reported as a primary or secondary finding is later reclassified (for example, from VUS to pathogenic/likely pathogenic, or from pathogenic/likely pathogenic to VUS, likely benign, or benign), an updated statement is issued to the original ordering healthcare provider at no additional cost.

Whole Exome Sequencing (WES), by covering all protein-coding genes of the genome, enables the detection of variants that are not related to the primary indication for testing but may have clinical relevance for patient care. These are referred to as secondary findings.

The reporting of secondary findings is performed in accordance with the recommendations of the American College of Medical Genetics and Genomics (ACMG) for the identification and disclosure of clinically actionable variants in specific genes.

Analysis and reporting of secondary findings are carried out only if prior consent (opt-in) has been provided by the patient or their legal guardian.

In cases where additional family members (e.g., parents) are included in the analysis, each individual is given the option to independently opt in for the analysis and reporting of secondary findings.

Secondary findings are presented in a separate section of the report and, where applicable, are reported individually for each tested person, independently of the findings reported for the primary (index) patient.