Exome sequencing

Exome sequencing

The next generation sequencing (NGS) method restricted to protein-coding regions of the genome. Covers 1% of the genome, but contains about 85% of disease-causing nucleotide sequence variants.

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Technical overview

Exome sequencing is a targeted, next generation sequencing (NGS) method that is restricted to the areas of the genome that encode protein. Exome covers approximately 1% of the genome but contains about 85% of the disease-causing nucleotide sequence variants.

For bioinformaticians trying to identify genes involved in more than 6,800 rare diseases among the plethora of findings, exome sequencing allows rapid and cost-effective identification of single-nucleotide polymorphisms and indels (small insertions and deletions).

Both inherited and de novo mutations can be identified, which may explain the heritability of Mendelian and complex disorders. In some cases, exome analysis can detect copy number variation (CNV).

1%of the genome is covered by the exome

85%of nucleotide sequence variants

6800rare diseases

In diagnoses with clinical heterogeneity

Examples: epilepsy, epileptic encephalopathies, muscular dystrophies/muscle disorders, ataxia, neuropathies, cardiomyopathies, connective tissue dysplasias, immunodeficiencies, deafness, blindness when major syndrome-associated variants are excluded

Atypical clinical manifestations or phenotypes. Clinically, it is difficult to isolate a specific gene or group of genes

Example: Hereditary ataxias, atypical parkinsonism

In genetically heterogeneous conditions where there's a high number of genes involved in the disease

Examples: mental retardation and severe immunodeficiency, autism

In the case of problematic heredity, when other genetic tests have failed to establish a diagnosis

Example: patient with neuropsychiatric retardation and similarly affected siblings, with a negative chromosomal micro-matrix analysis.

On the importance of clinical information

Different variants can be found during the interpretation of whole exome sequencing data - known pathogenic or benign, with unknown clinical significance or rare variants not previously described. In order to determine clinical significance and to match the variant found to a genetic disease, it is necessary to have as much clinical information about the proband and his or her immediate family members as possible. Withholding any clinical information, including the patient's family history, may affect the test results and their interpretation. Lack of clinical information may lead to exclusion of genetic variants that may be relevant to the patient and the assignment of incorrect clinical significance to identified variants.

DNA isolation → DNA library preparation → Region enrichment → Sequencing → Bioinformatics data processing


Includes sequence analysis of the entire exome of the patient, the parents or other family members.

The triple-pronged approach in exome sequencing improves diagnostic accuracy by facilitating the analysis of nucleotide sequence variants and enabling the detection of de novo mutations that underlie many diseases with early manifestation and severe course.

«Clinical exome»

Sequencing exons of only those genes whose mutations lead to the development of known diseases

More than 4,700 such genes have been described in the OMIM database so far. Although this type of analysis is cheaper, the list of genes is constantly being updated and is therefore less informative.

Panel sequencing

Based on the analysis of a group of genes combined into multigene panels. This type of diagnosis focuses on a specific syndromic indication.

Randomised results are unlikely with this approach. Panels go out of use as new genes associated with the disease are discovered or atypical symptoms are identified that overlap with indications for the use of a particular panel.

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A once-in-a-lifetime examination

As data is added, only re-analysis is required, not re-sequencing

Effective diagnosis

In epilepsy, congenital muscular dystrophy, dementia, prenatal testing and neonatal exome sequencing

6800 rare diseases

Identifies both inherited and de novo mutations

Scientific evidence

Diagnostic efficiency of exome sequencing


Epilepsy is the common denominator of a heterogeneous group of epileptic disorders with different causes, including genetics, and symptoms.

Difficulties in identifying epilepsies without diagnostic hypotheses with non-specific symptoms are quite common. The diagnostic efficacy of exome sequencing in patients with epilepsy is variously reported to be between 34 and 49% (Tumienė, B., et al.,2018, Forman E.B., 2018, Helbig KL., et al., 2016) compared to 8% when chromosome microarray analysis is used (Kozhanova T.V., 2019). Exome sequencing is 1.9 times more effective than the diagnostic output of the Hereditary Epilepsies panel (diagnostic efficiency 19.0%; P=0.0018)(Costain G., 2019). Establishing a genetic diagnosis can influence treatment choice, as has been demonstrated in patients with abnormalities in the SCN1ASCN8ASLC2A1 genes (Snoeijen-Schouwenaars, F. M., et al., 2018). 

To date, genetic testing in epilepsy is recommended to start with sequencing of a few genes and/or target panels and finally perform exome sequencing in the remaining undiagnosed cases.

Congenital muscular dystrophies (CMDs)

Congenital muscular dystrophies (CMDs) are a heterogeneous group of disorders characterised by a predominantly early childhood or adolescence onset, with signs of dystrophy primarily in the proximal muscles of the upper and lower extremities. The frequency of genetic diagnosis of CMD remains low, at around 24%. Families with undetected pathogenic variants of CMD face uncertainty about the risk of disease progression as well as transmission to offspring and the need for continued medical monitoring. The exomic genetic diagnosis rate of patients with muscular dystrophy, for whom targeted Sanger sequencing failed to identify the genetic cause of the disorder, was 40% for single probands and 34% to 60% for the ''trio'' sequencing (Ghaoui R., et al., 2015; Harris, E., et al., 2017).


Dementia is the most common neurodegenerative disorder, affecting between 1% and 3% of the population worldwide. Large genetic heterogeneity is challenging to diagnose, as the phenotype in many patients is either not syndromic or the molecular genetic cause is still unknown.

Exome sequencing is the gold standard in the diagnosis of neurodegenerative disorders. According to literature, exome sequencing has a diagnostic efficacy between 22% and 27% in patients who could not be diagnosed after a panel study (E Chérot, et al.,2018; Bojan Zalar, et al., 2018).

The relevance of prenatal testing

Approximately 2-4% of pregnancies are complicated by significant foetal malformations. The prenatal testing strategy and choice of tests should be individualised and correlated with ultrasound findings and family history. Current options include: karyotype analysis, fluorescent in situ hybridisation (FISH) and chromosomal microarray analysis (CMA) for analysis of chromosomal abnormalities.

Various karyotypic methods provide a diagnostic efficiency of about 35% (Zhang S., 2017, Hillman S.C., 2013). Thus, when these techniques are used, more than half of fetuses with structural abnormalities go undiagnosed. 

Two recent major studies have reported the results of exome trio sequencing performed on fetuses with malformations and normal karyotype (Petrovski S., et al., 2019, Lord J., et al., 2019). Both studies show that exome sequencing increases the diagnostic yield of fetuses with congenital malformations by approximately 8-19% after karyotyping and CMA results, and the frequency of detection of pathogenic variants is strongly correlated with the number of fetal anomalies. 

Exome sequencing is a phenotype-dependent test, so the physician must provide the laboratory with adequate information necessary to obtain the most accurate interpretation of the results. Clinical information should include detailed reports of fetal magnetic resonance imaging (MRI) and/or fetal ultrasound, preliminary results of combined screening, ethnicity, reproductive history and family history, including parental consanguinity.

The «trio» analysis is preferable to the fetus-only or fetal and single parent analysis. The «trio» analysis has higher diagnostic coefficients (by 20%) (Retterer K., 2016).

If fetal abnormalities are significantly suggestive of a specific diagnosis, single gene testing or a phenotypic gene panel is more appropriate as a first-line test. Prenatal exome sequencing can only be performed if there is a clinically relevant indication for such a test. In the absence of such indications, interpretation of the data obtained is not possible.

Exome sequencing of newborns

According to WHO, 4-6% of newborns worldwide are born each year with severe congenital malformations. Congenital malformations are estimated to be present in 13% of all neonatal intensive care unit admissions in developed countries (Widmann R., et al., 2017) and remain the leading cause of neonatal mortality. A literature review shows that exome sequencing has a high diagnostic yield (36-48%) in the diagnosis of infants with very severe congenital malformations in the absence of molecular cytogenetic abnormalities (Trujillano, D., et al.,2017, Meng L., et al., 2017, QI Zhi-Ye., et al.,2019).

An atypical and previously undescribed form of genetic disorder seen in young children challenges the traditional paradigm of multilevel genetic testing in intensive care units. Targeted panels are reasonable in most cases, but failure or duration in detecting causative variants in critically ill children is a significant challenge that can be overcome by exome sequencing.

Having minimal clinical information, i.e. only one single symptom, reduces the specificity of the results. As it is difficult to detect the full clinical picture in the first days of life, it is recommended to do a 'trio' analysis only when standard diagnostic methods such as karyotype analysis, gene panels and CMA are not feasible.

Exome sequencing can detect some genetic changes that are not related to the patient's current signs and symptoms (secondary findings). Nevertheless, these findings may have important implications for the health of patients and their family members.

These disorders include:

  • some cancer syndromes,
  • connective tissue disorders,
  • certain types of cardiovascular disease,
  • high cholesterol levels and susceptibility to complications from anaesthesia.

On the other hand, some types of genetic disorders have no effective treatment and can lead to death or lifelong disability. Secondary findings may be included in the patient report.v

Secondary findings may not be included in the report if you notify us that you do not want to be informed of them.

Randomly found variants in genes

When interpreting randomly found variants in genes associated with diseases with incomplete penetrance and late manifestation, it is difficult to assess the risk to the patient. Similarly, it is important to consider the large number of variants identified in exome sequencing of patients, prioritising them for follow-up.

Features of the «trio» analysis

The «trio» exome sequencing may reveal the parent-child unrelatedness and the fact of a closely related marriage. The patient should also be informed about this.

NGS, or next generation sequencing

Next generation sequencing. The term means determining the nucleotide sequence (examining the primary structure) of DNA or RNA. The technology allows several sections of the genome to be 'read' at once. The size of a single fragment read varies from 25 to 500 base pairs.

Copy number variation (CNV)

A type of genetic polymorphism in which individual genomes differ in the number of copies of chromosomal segments ranging in size from 1,000 to several million base pairs.


Loss of a portion of the DNA sequence of the nitrogenous bases and loss of the corresponding nucleotides.

DNA library (genomic library)

A set of DNA fragments subjected to sequencing. These DNA fragments are flanked by identical DNA adapters – special sequences required for the simultaneous sequencing of many different DNA fragments.


A change in the sequence of DNA when an insertion of a DNA sequence occurs. The minimum size of such an insertion is one nucleotide

De novo mutations

A change in a DNA sequence that occurs for the first time in germ cells (gonads) or during fertilisation, as opposed to inherited.

Single-nucleotide polymorphism

Differences of one nucleotide (A, T, G or C) in a DNA sequence in the genome that occur in a population with a frequency of more than 1% (or in another comparable sequence) of members of the same species or between regions of homologous chromosomes.

Genome sequencing

Identification of the entire DNA sequence, including non-coding areas. Different from exome sequencing in this respect.

[1] Bojan Zalar, Aleš Maver, Ana Kovanda, Ana Peterlin & Borut Peterlin: CLINICAL EXOME SEQUENCING IN DEMENTIAS: A PRELIMINARY STUDY Psychiatria Danubina, 2018; Vol. 30, No. 2, pp 216-219.

[2] Widmann R, Caduff R, Giudici L, et al. Value of postmortem studies in deceased neonatal and pediatric intensive care unit patients. Virchows Arch. 2017;470(2):217-223.

[3] Costain G, Cordeiro D, Matviychuk D, Mercimek-Andrews S., Clinical Application of Targeted Next-Generation Sequencing Panels and Whole Exome Sequencing in Childhood Epilepsy. Neuroscience. 2019 Oct 15;418:291-310

[4] E Chérot, B Keren, C Dubourg, W Carré, M Fradin, et al.. Using medical exome sequencing to identify the causes of neurodevelopmental disorders: Experience of 2 clinical units and 216 patients. Clinical Genetics, Wiley, 2018, 93 (3), pp.567-576.

[5] Forman EB, Gorman KM, Conroy J, et alCost of exome sequencing in epileptic encephalopathy: is it ‘worth it’?Archives of Disease in Childhood 2018

[6] Ghaoui R, Cooper ST, Lek M, et al. Using whole exome sequencing to diagnose limb-girdle muscular dystrophy : results and lessons learned . JAMA Neurol. 2015;72(12):1424–1432.

[7] Harris, E., Topf, A., Barresi, R. et al. Exome sequences versus sequential gene testing in the UK highly specialised Service for Limb Girdle Muscular Dystrophy. Orphanet J Rare Dis 12, 151 (2017).

[8] Helbig KL, Farwell Hagman KD, Shinde DN, et al. Diagnostic exome sequencing provides a molecular diagnosis for a significant proportion of patients with epilepsy. Genet Med 2016;18:898-905.

[9] Hillman SC, McMullan DJ, Hall G, et al. Use of prenatal chromosomal microarray: prospective cohort study and systematic review and meta-analysis. Ultrasound Obstet Gynecol. 2013;41:610–620.

[10] Lord J, McMullan DJ, Eberhardt RY, et al. Prenatal exome sequencing analysis in fetal structural anomalies detected by ultrasonography (PAGE): a cohort study. Lancet. 2019;393:747–757.

[11] Meng L, Pammi M, Saronwala A, et al. Use of Exome Sequencing for Infants in Intensive Care Units: Ascertainment of Severe Single-Gene Disorders and Effect on Medical Management. JAMA Pediatr. 2017

[12] Petrovski S, Aggarwal V, Giordano JL, et al. Whole-exome sequencing in the evaluation of fetal structural anomalies: a prospective cohort study. Lancet. 2019;393:758–767.

[13] Retterer K, Juusola J, Cho MT, et al. Clinical application of whole-exome sequencing across clinical indications. Genet Med. 2016;18:696–704.

[14] Snoeijen-Schouwenaars, F. M., van Ool, J. S., Verhoeven, J. S., van Mierlo, P., Braakman, H. M. H., Smeets, E. E., … Willemsen, M. H. (2018). Diagnostic exome sequencing in 100 consecutive patients with both epilepsy and intellectual disability. Epilepsia.

[15] Trujillano, D., Bertoli-Avella, A., Kumar Kandaswamy, K. et al. Clinical exome sequencing: results from 2819 samples reflecting 1000 families. Eur J Hum Genet 25, 176-182 (2017)

[16] Tumienė, B., Maver, A., Writzl, K., Hodžić, A., Čuturilo, G., Kuzmanić-Šamija, R., (2018). Diagnostic exome sequencing of syndromic epilepsy patients in clinical practice. Clinical Genetics, 93(5), 1057–1062.

[17] QI Zhi-Ye, DUAN Jiang,HE Xiang-Ying et al. Clinical application of whole exome sequencing in monogenic hereditary disorders in critically ill newborns[J]. CJCP, 2019, 21(7): 640-643.

[18] Zhang S, Lei C, Wu J, et al. A retrospective study of cytogenetic results from amniotic fluid in 5328 fetuses with abnormal obstetric sonographic findings. J Ultrasound Med. 2017;36:1809–1817.

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Single-nucleotide polymorphisms

Differences of one nucleotide (A, T, G or C) in a DNA sequence in the genome that occur in a population with a frequency of more than 1% (or in another comparable sequence) of members of the same species or between regions of homologous chromosomes.

De novo mutations

A change in the DNA sequence that occurs for the first time in germ cells (gonads) or during fertilisation, as opposed to inherited.