What is whole exome sequencing (WES)?

Overview

The human genomeMolecules like DNA and RNA. has approximately three billion base pairs; only one to five percent are translated into significant proteins. Any mutations that occur in these proteins result in phenotypic consequences.

We can use WES to find these malfunctions in the genome.

We define exons as the coding part of the DNA that takes part in the formation of proteins. These exons comprise approximately one to five percent of the genome and are collectively known as the exome.

The majority of the disease-causing variants occur in the exome. The exome is one of the best-understood parts of the human genome; thus, its sequencing makes detecting diseases easier.

Methodology

We can define WES as:

An efficient and comprehensive genetic test that identifies the DNA changes rapidly and reliably.

It's a genomic technique for sequencing all protein regions of a genome. The basic gist of the WES working comprises two parts. These are:

1. Select only those parts of the DNA that encode the proteins or the exons.
2. Sequence the exonic DNA using any high throughput DNA sequencing technology.

Step 1: Strategies for target enrichment

Target enrichment strategies allow us to selectively capture the genomic regions that are required from DNA.

There are many techniques for target capturing the DNA; only a few can capture the entire exome. There are two techniques for this:

Array-based capturing

Single-stranded oligonucleotidesShort, synthetic strands of the DNA or RNA. are captured from the genome with the region of interest displayed on the surface. This requires microarrays from which oligonucleotides are extracted.

DNA is then sheared to form double-stranded structures. These are then passed through the end repairing process, and universal priming sequencesComplementary sequences to the nucleotide sequences. These are very common in some DNA molecules. are added. These fragments are then hybridized. Unhybridized parts are washed off, and desired parts are acquired. These fragments are then amplified using any one of the following methods:

• Polymerase chain reaction (PCR)
• Sequence capture human exome 2.1M array
• Agilent capture array
• Comparative genomic hybridization array

In-solution capturing

A pool of custom oligonucleotides is synthesized and hybridized to fragment the DNA sample. The oligonucleotides act as probes and are labeled with beads. These selectively hybridize the genomic regions of interest, whereas the excess parts are washed away. The beads are then removed, and genomic sequences are then sequenced selectively.

The illustration above shows how the DNA sequences are ligated to the oligonucleotides. Then the hybridization process takes place, and the labeling of beads is carried out. After capturing the probes, the selected regions are ready for next-generation sequencing.

Step 2: Sequencing of exons

There are many next-generation sequencing platforms available for the sequencing of exons. Some of these are:

• Sanger sequencing methodologies
• Roche 454
• Life Technologies SOLiDSequencing by Oligonucleotide Ligation and Detection systems
• Life Technologies Ion Torrent
• Illumina genome analyzer II
• NovaSeq series instruments

All these are used for analyzing relatively short stretches of DNA sequences.

Advantages of WES

Following are the advantages of WES:

• Since the exome has a fractional part of the genome, it is sequenced quickly and easily.
• Quick sequencing results in a faster diagnosis of the disease.
• Since most of the disease-causing variants are found in the exome, the results influence the management and treatment results.
• WES allows a comprehensive coverage of the exons to target medically relevant genomic regions, including the disease-associated sites and the untranslated regions.
• It provides a cost-effective solution as compared to WGSWhole Genome Sequencing as the whole genome is not to be sequenced.
• Next-generation sequencing technology allows the increased variant discovery potential as low frequency and rare mutations are discovered.
• It produces a manageable dataset that is easy to analyze.

Conclusion

WES, along with exome enrichment, can effectively and efficiently help in identifying the coding variants. These variants include a broad range of applications, including the study of cancer, the genetics of the population, and genetic diseases.