When Should Whole Exome Sequencing Be Considered?
Not every diagnostic challenge is caused by a lack of genetic testing.
Quite often, the issue is that the most appropriate genetic tests have not been performed for the clinical situation at hand.
Although → Whole Exome Sequencing (WES) is now one of the most comprehensive diagnostic tools available in medical genetics, there are several situations in which its use is particularly appropriate:
• when a rare disease or genetic syndrome is suspected, but its precise cause remains unclear;
• when symptoms, clinical findings, malformations, or functional abnormalities involve multiple organs or body systems;
• when different specialists have proposed different diagnostic hypotheses;
• when previous genetic panels have failed to provide a conclusive answer;
• when the diagnostic journey continues without a unifying explanation.
In these situations, Whole Exome Sequencing allows the simultaneous analysis of the entire coding region of the human genome, significantly expanding the available diagnostic space.
It is important not to confuse Whole Exome Sequencing with so-called Clinical Exome Sequencing, which limits the analysis to genes currently known to be associated with disease (approximately 6,000 genes). Although the two terms are sometimes used interchangeably, they refer to different approaches with different diagnostic potential.
The term Clinical Whole Exome Sequencing may instead be used to emphasize the clinical diagnostic application of an analysis that is nevertheless performed across the entire exome.
The Exome Is Not Always the Same
When discussing → Whole Exome Sequencing, it is often considered a single, standardized test. In reality, several different testing configurations are available, and these can significantly influence the effectiveness of the analysis.
In its simplest form, only the patient’s DNA is analyzed (Singleton WES).
In other situations, both biological parents are analyzed simultaneously (Trio WES), allowing direct comparison of genetic variants within the family. Occasionally, particularly in hospital-based settings, additional family members may also be included. For example, an affected or unaffected sibling of the index case (the first affected child in the family) may be added to the analysis (Quad Exome Sequencing). In even more specific situations, sequencing may be performed exclusively on two siblings or other related individuals (Duo Exome Sequencing).
The choice between Singleton WES and Trio WES depends on several factors, including the patient’s age, the clinical suspicion, the availability of family members, and the objectives of the diagnostic investigation.
Particularly in children with a suspected genetic disorder, Trio WES can increase the likelihood of identifying causative variants and facilitate the interpretation of the results.
What Happens After Exome Sequencing?
Whole Exome Sequencing may represent the final step of a diagnostic journey or, in some cases, one of its most important milestones.
In some patients, the analysis rapidly identifies a causative variant, leading to a definitive molecular diagnosis. Not infrequently, particularly after years of diagnostic uncertainty, this represents not only an answer and a relief, but also a reinterpretation of an entire period of life.
In other cases, variants are identified that require additional clinical, familial, or functional investigations.
Sometimes, however, the result is negative. This does not necessarily mean that a genetic cause has been excluded. Some pathogenic variants may belong to categories that cannot be reliably detected by exome sequencing, while others may lie outside coding regions, requiring alternative technologies or a broader analytical approach (for example → Whole Genome Sequencing).
In summary, regardless of the outcome, Whole Exome Sequencing — whether performed as a singleton or trio analysis — represents a fundamental diagnostic step and has become an essential component of the evaluation of many complex genetic conditions.
→ If this situation sounds familiar, the diagnostic pathway can start here.
To analyze all 20,000 human genes simultaneously
Exome Sequencing refers to the sequencing of the coding regions (exons) of an individual’s genes.
It is important to distinguish between Whole Exome Sequencing (WES), which includes all approximately 20,000 human genes, and Clinical Exome Sequencing (CES), which is limited to genes currently known to be associated with disease (approximately 6,000 genes).
An even greater distinction exists between WES, CES, and gene panels based on the same sequencing technologies but restricted, at the bioinformatic and clinical interpretation level, to a predefined subset of genes.
History
For many years, the traditional genetic test consisted of sequencing a single gene (or a small panel of genes) using capillary electrophoresis, more commonly known as Sanger sequencing.
The advent of Next Generation Sequencing (NGS) technologies has transformed the study of human genetic disease, ushering in an era of genomic data generation unprecedented in both scale and speed.
Thanks to the continuous optimization of NGS technologies, it has become possible to analyze an extraordinarily large number of DNA fragments simultaneously, enabling the sequencing of thousands of genes in parallel or, in the case of exome sequencing, all approximately 20,000 human genes, corresponding to the entire coding portion of the genome.
This has had a profound impact on both routine clinical diagnostics and biomedical research.
Although the coding region represents only about 1% of the human genome, it is estimated that up to 85% of known pathogenic variants are located within these regions. The progressive adoption of → Whole Genome Sequencing (WGS) has made it possible to identify disease-causing variants located outside coding regions. Nevertheless, Whole Exome Sequencing (WES), whether performed as a singleton or trio analysis, continues to demonstrate remarkable diagnostic performance and still offers one of the best cost-to-diagnostic-yield ratios among high-throughput genomic technologies.
The introduction of Copy Number Variant (CNV) analysis into modern Whole Exome Sequencing workflows has further expanded the diagnostic potential of exome testing. Today, in most advanced clinical genomics laboratories, CNV analysis is routinely included as part of standard exome interpretation.
This has significantly changed the diagnostic approach to many genetic conditions, particularly in patients with developmental delay, intellectual disability, and multiple congenital anomalies. In the past, diagnostic workflows often relied on karyotyping and chromosomal microarray analysis (array-CGH) as first-tier tests, reserving exome sequencing for later stages of investigation.
By allowing the simultaneous detection of both sequence variants and many copy number alterations, Whole Exome Sequencing is increasingly considered a first-line genomic test. In many cases, genomic alterations traditionally investigated by array-CGH can also be detected through exome-based CNV analysis, sometimes at even higher resolution in adequately covered regions.
For this reason, current trends in clinical genomics increasingly position Whole Exome Sequencing among the primary diagnostic tools for complex genetic disorders.
The End of the Diagnostic Odyssey
For many years, Whole Exome Sequencing (WES) was considered a second- or third-tier test, reserved for patients in whom numerous previous investigations had failed to establish a diagnosis.
Today, particularly in more advanced healthcare systems, this paradigm is progressively changing. Thanks to its high diagnostic yield and its ability to interrogate the entire coding portion of the human genome simultaneously, Whole Exome Sequencing is increasingly being adopted as a first-line test in complex genetic conditions, ranging from rare pediatric disorders to adult-onset neurological diseases.
According to numerous studies, patients affected by rare diseases often wait years before receiving a definitive diagnosis. Historically, the average diagnostic journey could exceed seven years, and cases extending over several decades are not uncommon.
This situation, characterized by repeated specialist consultations, inconclusive investigations, and frequent changes in diagnostic hypotheses, is commonly referred to as the diagnostic odyssey.
Today, Whole Exome Sequencing, whether performed as a singleton or trio analysis, together with Whole Genome Sequencing (WGS), represents one of the most effective tools for shortening or interrupting this process by concentrating, into a single test, information that previously required years of sequential investigations.
As previously discussed, the term exome sequencing is sometimes used to describe very different types of analyses. In some cases, even panels restricted to a few hundred genes are incorrectly referred to as “exomes”.
The most important distinction remains that between Clinical Exome Sequencing (CES) and Whole Exome Sequencing (WES).
Clinical Exome Sequencing limits the analysis to genes currently known to be associated with disease (approximately 6,000–7,000 genes), whereas Whole Exome Sequencing covers the entire coding region of the human genome, comprising approximately 20,000 genes and often including dedicated coverage of mitochondrial DNA (mtDNA).
In modern rare disease diagnostics, the role of Clinical Exome Sequencing has progressively declined in favor of Whole Exome Sequencing, which offers greater analytical completeness, improved opportunities for future data reanalysis, and greater long-term interpretative flexibility.
For this reason, when the goal is to identify the genetic cause of a complex condition, Whole Exome Sequencing is generally considered the preferred approach.
Breda Genetics Exome Portfolio
Breda Genetics offers one of the most comprehensive Whole Exome Sequencing portfolios available for clinical use. As specialists in rare diseases and molecular geneticists with extensive diagnostic experience, we have learned to apply exome sequencing across a wide range of clinical and research settings.
All our Whole Exome Sequencing solutions are based on sequencing the entire coding region of the human genome, encompassing approximately 20,000 genes.
Available both as a clinical diagnostic service and as a sequencing-only solution for partner laboratories, our current platforms include Twist Exome RefSeq + mtDNA and Agilent SureSelect Human All Exon V8.
Coverage depth remains consistently high and is supported by rigorous quality standards throughout the analytical process. High-quality alignments, optimized bioinformatic workflows, and extensive internal reference datasets also enable increasingly accurate detection of Copy Number Variants (CNVs), now an integral part of modern genomic interpretation.
Applications of Exome Sequencing
1. Diagnosis of Mendelian Disorders
Mendelian disorders represent one of the primary applications of Whole Exome Sequencing. Before the advent of modern genomic technologies, identifying the gene responsible for a rare disease often required complex family studies, extensive genomic mapping, and years of experimental work.
Today, Whole Exome Sequencing enables simultaneous analysis of the entire coding region of the genome, allowing direct identification of pathogenic or likely pathogenic variants across thousands of disease-associated genes.
This technological revolution has profoundly transformed medical genetics. Numerous disease genes have been identified through Whole Exome Sequencing, and studies based on both Whole Exome Sequencing and Whole Genome Sequencing continue to expand our understanding of genetic disease.
Every year, new gene–disease associations and new clinical manifestations of previously known genes (phenotype expansion) are described, contributing to improved diagnosis and understanding of rare disorders.
2. Genomic Research and Complex Diseases
Beyond the diagnosis of rare Mendelian disorders, both Whole Exome Sequencing and Whole Genome Sequencing have significantly contributed to the understanding of complex diseases. The identification of new genetic variants and biological mechanisms continues to expand our knowledge of common conditions, including cardiovascular, neurological, and immunological disorders.