A child with multiple symptoms.
Sometimes also malformations.
Different systems involved.
No single pattern that explains everything.
Biochemical tests, imaging, and other investigations have already been performed.
Some results are normal.
Others suggest possible directions, but a unifying diagnosis remains elusive.
At this stage, high-throughput sequencing enters the picture.
Most often, pediatricians and neuropsychiatrists recommend → Whole exome sequencing (WES).
A further question then arises:
how should → whole exome sequencing be performed?
Whole exome sequencing exists in different configurations.
In some cases, this choice directly shapes the interpretation and diagnostic yield.
WES trio to support interpretation
WES trio refers to the analysis of the patient together with both biological parents.
This allows genetic findings to be interpreted within a familial context, making it easier to understand how variants relate to each other.
When WES trio becomes relevant
WES trio is considered in clinical situations where interpretation requires higher resolution.
This includes:
- complex or multisystem presentations
- early-onset conditions in children
- cases where inheritance patterns are critical for interpretation
In selected clinical contexts, WES trio may increase the diagnostic yield,
often from around 30–35% in singleton analysis up to approximately 45% with a trio approach.
→ Whole exome sequencing
In these situations, the difference does not lie in performing the test.
It lies in how the test is done from the beginning.
→ If this situation feels familiar, start the diagnostic pathway here.
Singleton vs trio exome sequencing
→ Whole exome sequencing can be performed on a single individual or as a trio.
In a singleton approach, interpretation is based solely on the patient’s data.
In a trio approach, the inclusion of both biological parents introduces an additional layer of context.
This directly influences:
- the identification of de novo variants (variants arising in the patient and not present in the parents)
- the interpretation of inheritance patterns (e.g. faster recognition of compound heterozygosity)
- the dismissal of certain VUS (variants of uncertain significance excluded based on inheritance from a healthy parent)
Diagnostic yield of WES trio
In selected clinical contexts, WES trio can significantly increase the diagnostic yield.
In prenatal settings, particularly in fetuses with ultrasound anomalies, trio sequencing may increase the diagnostic rate by up to +15% beyond what is achieved with karyotyping and chromosomal microarray.
In children with syndromic and non-syndromic neurodevelopmental disorders, the cumulative diagnostic yield of WES trio — including both SNVs and CNVs — can approach ~50%.
This reflects the added value of familial context, rather than differences in sequencing technology.
WES trio is considered in clinical situations where interpretation benefits from the cross-analysis of the patient and both biological parents.
This includes:
- complex or multisystem presentations (sometimes referred to as polysyndromic conditions), where the diagnosis remains particularly elusive
- early-onset conditions in children, especially when the clinical course is not clearly defined and requires NICU-level interventions
- cases in which inheritance patterns guide interpretation, and distinguishing between inherited and de novo variants becomes essential
- cases where the clinical suspicion may appear clear, but the genetic heterogeneity or extreme rarity of the condition makes a panel or single-gene approach statistically inefficient or technically unfeasible
In these settings, the inclusion of parental data directly improves the clarity of the result and can significantly accelerate the diagnostic process and subsequent clinical decisions.
How WES trio is performed
WES trio requires DNA samples from the patient and both biological parents.
Sampling can be performed in different ways, depending on the clinical context:
→ Blood sampling at home with a dedicated nurse
→ In-clinic sampling during consultation (buccal swab) — particularly useful in very young patients, where blood collection may be more challenging
→ Self-collection using a dedicated buccal swab kit
In some cases, extended family-based approaches may be considered, such as sequencing additional siblings (WES tetra).
These configurations can further support interpretation in selected situations.
All samples are processed within a clinical diagnostic framework, where sequencing and interpretation are integrated from the beginning.
This ensures that results are not only generated, but immediately interpreted in a clinically meaningful way.
Choosing how exome sequencing is performed
Whole exome sequencing is often perceived as a single test.
In practice, the way it is performed from the beginning can shape the entire diagnostic process.
In selected clinical situations, choosing a trio approach rather than a singleton analysis allows for a more structured interpretation from the start.
This does not change the technology.
It changes how the result is understood, and how quickly a diagnosis can be reached.