Whole exome sequencing

Whole exome sequencing (WES) is a next-generation sequencing (NGS)-based test in which the protein-coding regions of all of a patient’s genes (known as the exome) are tested simultaneously. The exome makes up around 1% of a patient’s genome. Non-coding regions of the genome are not tested, and the range of variant types detected is slightly smaller than for whole genome sequencing (WGS).

It is important to be aware that virtual panels of genes may be used in clinical applications of WES. This means that even though all genes are sequenced, only those genes known to be associated with the patient’s features are analysed. Therefore, it’s important to remember that just because you have requested a test that is described as a WES panel test, this does not usually mean that all of your patient’s genes have been checked – just those that are included on the panel.

Alternatively, a gene-agnostic (looking at all genes), family-based approach can be used. This may be appropriate to find a diagnosis for patients whose condition is suspected to have a genetic cause but the specific causative genes are unknown. This is particularly effective when a child is tested alongside parents (trio sequencing) and variants are filtered according to their inferred inheritance patterns.

WES can sometimes give results faster than WGS. This is why one of the current clinical applications in the NHS is for the rapid diagnosis of acutely unwell children.

In addition, WES has proven to be a useful research tool, particularly for screening large cohorts of patients with unexplained rare disease for novel causal genes. An example of this is the Deciphering Developmental Disorders (DDD) study, a UK-wide study that undertook WES and genome-wide microarray in children with developmental disorders. The WES data were first analysed for variants in more than 1,000 genes known to cause developmental disorders, giving a high diagnostic yield in previously undiagnosed children. For children who remained undiagnosed, genes with no existing association with developmental delay were also analysed, resulting in the identification of several novel causal genes.

WES is done using short-read NGS technology. Briefly, patient genomic DNA is fragmented and the DNA representing protein-coding regions is enriched during laboratory processing. Sequencing data are generated only for these protein-coding regions  and a small amount of adjacent non-coding DNA. Data analysis may then be restricted to a subset of genes relevant to the patient’s features using a virtual panel. All data are stored.    

Advantages

WES can be used to test a wide range of genes simultaneously and can be faster and more cost effective than WGS. Some of the advantages are outlined below.

• Single nucleotide variants and small insertions and/or deletions are detected with a high accuracy.
• Copy number variants may be detected (but potentially with lower accuracy than in WGS).
• In WES, all of an individual’s genes are sequenced. This means that where an initial analysis does not yield a diagnosis, it may be possible to go back to the original data in the future to consider newly discovered causal genes. Development of policy on whether and when NHS labs will provide reanalysis of WGS and WES data is still in progress.
• WES is less expensive than WGS, but can be more expensive than a targeted gene panel.
• WES may generate fewer variants of uncertain significance (VUS) than WGS.
• WES is particularly powerful in identifying the genetic cause of rare disease in a child when parents are tested alongside (trio exome sequencing).
• In research applications, WES can be used to identify novel genetic causes of disease.

Limitations

• When WES is used for the diagnosis of rare disease patients, it is important to be aware that virtual panels are often used. This means that, although sequencing data are generated for all genes, only genes known to be associated with the patient’s features are analysed. In contrast, if a gene-agnostic approach to analysis is chosen, WES can detect variants in protein-coding regions of all of a patient’s genes.
• Clinical interpretation of the large number of identified variants can be a challenge.
• More VUS are generated compared to more targeted testing.
• There is an increased risk of incidental findings compared to more targeted testing.
• There are regions of the genome that pose a technical challenge for short-read sequencing, the type of sequencing currently used for most diagnostic WES. These regions, which can include those with pseudogenes or those that contain repetitive elements, may therefore not be analysed.
• Mosaicism may not be detected, as the read depth used is often limited.
• WES will not detect variants in non-coding regions (unlike WGS), with the exception of canonical splicing regions immediately next to a coding region.
• WES may not detect copy number variants, and will not detect structural rearrangements (unlike WGS).

You can view a summary table comparing the advantages and disadvantages of the different approaches to gene sequencing (gene panel, clinical exome, WES, WGS).

• WES only sequences the protein coding regions of the genome.
• Virtual panels are often applied to WES data, which means that all the data may not be analysed.
• Gene agnostic analysis looks at the data from all the genes sequenced and does not apply virtual panels.

For clinicians

• Deciphering Developmental Disorders study

References:

• Fitzgerald TW, Gerety SS, Jones WD and others. ‘Large-scale discovery of novel genetic causes of developmental disorders‘. Nature 2015: volume 519, issue 7,542, pages 223–228. DOI: 10.1038/nature14135
• Seaby EG, Pengelly RJ, Ennis S. ‘Exome sequencing explained: a practical guide to its clinical application‘. Briefings in Functional Genomics 2016: volume 15, issue 5, pages 374–384. DOI: 10.1093/bfgp/elv054
• Taylor A, Alloub Z and Tayoun AA. ‘A simple practical guide to genomic diagnostics in a pediatric setting‘. Genes (Basel) 2021: volume 12, issue 6, page 818. DOI: 10.3390/genes12060818
• Williamson SL, Rasanayagam CN, Glover KJ and others. ‘Rapid exome sequencing: revolutionises the management of acutely unwell neonates‘. European Journal of Paediatrics 2021: volume 180, issue 12, pages 3,587–3,591. DOI: 1007/s00431-021-04115-x
• Wright CF, Fitzgerald TW, Jones WD and others. ‘Genetic diagnosis of developmental disorders in the DDD study: a scalable analysis of genome-wide research data‘. The Lancet 2015: volume 385, issue 9,975, pages 1,305–1,314. DOI: 10.1016/S0140-6736(14)61705-0