Clinical whole exome sequencing (WES)

Sequence analysis of all known human genes (whole exome sequencing – WES), through Next Generation Sequencing technologies (NGS) is the new genetic super- weapon, as it is theoretically in a position to uncover the molecular basis for any genetic disorder in an affected individual, which has not been diagnosed by other available conventional genetic testing options.

This test is completely different from other types of currently known genetic testing, in terms of the number of genes that are analyzed simultaneously. Until now, classical approaches to the diagnosis of genetic diseases involved the investigation of specific targeted genes (1-5), considered to be associated with the observed phenotype.


Today, this new and powerful technology allows high-throughput analysis of all ~22,000 human genes (whole exome sequencing) or, alternatively, the simultaneous analysis of hundreds of genes (gene panels) associated with different categories of diseases with a genetic etiology.


Main concepts

Exome = the coding part of our genome = all known human genes. It includes ~22.000 genes, >160.000 exons, covers ~40-50 Mb (40-50 million bases) and includes the analysis of intron-exon junctions (splice sites).

Why perform genomic analysis – whole exome sequencing

Currently, whole exome sequencing is useful:

  • for uncovering pathogenic mutations, for rare (or complex common) genetic disorders
  • for identifying pathogenic mutations, causing or predisposing for common genetic diseases, e.g. cancers, intellectual disability, autism
  • for detecting somatic mutations (mosaicism)

Today, whole exome sequencing is not so useful:

  • for identifying common variants/polymorphisms with unknown functional significance
  • for personal genomic profiling or for communicating predispositions and risks for common diseases

When do we apply whole exome sequencing

The application of this highly specialized test is aimed at clinicians and their patients, who suffer from an as yet undiagnosed disease believed to have a genetic etiology. This does not mean that there must necessarily exist a prior family history of the disease, since in the case of recessive or sporadic disorders (which constitute the majority of genetic diseases) there is usually only one affected family member.

Based on the published data up to date, the application of whole exome sequencing has proven extremely useful for diagnosing the cause of neuro-developmental pediatric diseases, complex syndromes, undiagnosed neuromuscular disorders, autism, etc., and is particularly valuable in the management of oncology cases-cancers.

Due to the fact that we still do not understand the precise function of many human genes, as well as their interactions, it is possible to fail to reach a definitive diagnosis, especially in cases with ill-defined clinical symptoms.

How is clinical whole exome sequencing performed and how long does the test take to be completed

We perform whole exome sequencing (WES), via Next Generation Sequencing (NGS) on a Genome Analyzer – Ion Proton platform.

As a first step, the patient’s DNA is subjected to standard QC procedures, in terms of size distribution and exact DNA quantity. The applied methodology aims at analyzing the DNA sequence of the exons of all known human genes (CCDS genes), as represented in the Ion Ampliseq Exome Sequencing reagent.

Bearing in mind that the vast majority of pathogenic mutations (>80%) are located in the coding regions – exons of the genes and the exon-intron boundaries, as opposed to deep intronic mutations which are typically non-pathogenic, the technique of whole exome sequencing (WES) permits the simultaneous identification of pathogenic mutations in almost all known human genes related to genetic diseases.

However, it must be noted that the technique, as applied today, is in a position to analyze successfully the DNA sequence of approximately 90-95% of all exons (CCDS), as compared to the Reference Genome (NCBI hg19).

Furthermore, the depth of read and coverage is not the same across all regions, and therefore the confidence of variant calling may vary.

DNA sequence alignment and variant calling is performed with the aid of the Ion Torrent Server Suite, Ion Reporter, Softgenetics NextGene v2.1 and DNAnexus analysis tools.

All genomic variant data are imported, filtered and prioritized using an in-house proprietary Exome Analysis Application (EMA®) software pipeline and novel mutations are assessed for pathogenicity through the pipeline, utilizing a combination of several in silico prediction tools, with reference to numerous public databases and resources and an internal Greek variant frequency database of 120 individuals.

Finally, the test generally evaluates and reports:

  • known pathogenic mutations, previously reported in affected individuals and which have been directly linked to the expression of the disease, or
  • novel obligatory pathogenic variants/mutations, such as nonsense, frameshift, canonical splice-site variants, indels.

In certain cases, it is possible that other type of variants, not included in any of the above categories, may also be evaluated and reported.

The applied methodology has certain important limitations:

  • It may not detect variants in ‘blind spots’ of exons, which may have not been analyzed in the sample (typically <10%) or may be generally refractory to this type of analysis (e.g. highly GC-rich regions, etc.).
  • It is not able to reliably detect extensive genomic deletions/duplications (copy number variants).
  • It is not in a position to detect triplet-repeat expansions, associated with a certain type of genetic disorders ( triplet-repeat disorders).
  • The evaluation and reporting adheres in general to the ACMG guidelines (Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med. 2015;17:405-24).

The test results are typically available within 4-6 weeks, but this may vary depending on the clinical findings of each patient.

The test is highly sensitive and complex. Therefore, the analysis and the clinical evaluation of the results should be performed by a highly skilled group of clinical and molecular geneticists, ensuring the maximum diagnostic validity for the patient and the family.

Proper clinical genetic assessment and genetic counseling, both before and after testing, is essential in order to determine the optimum testing strategy and also to communicate properly the concepts of pathological and normal.

The test is usually completed in 4-6 weeks

InterGenetics is a Ion Torrent™ Certified Service Provider for Ion AmpliSeq sequencing on the Ion Proton platform.