Elsevier

The Lancet Neurology

Volume 10, Issue 10, October 2011, Pages 942-946
The Lancet Neurology

Rapid Review
Exome sequencing: a transformative technology

https://doi.org/10.1016/S1474-4422(11)70196-XGet rights and content

Summary

Background

Much basic research into disease mechanisms has made use of genetic findings to model and understand aetiology. Broad success has been achieved in finding disease-linked mutations with traditional positional cloning approaches; however, because of the requirements of this method, these successes have been limited by the availability of large, well characterised families. Because of these and other restrictions the genetic basis of many diseases, and diseases in many families, remains unknown.

Recent developments

Exome sequencing uses DNA-enrichment methods and massively parallel nucleotide sequencing to comprehensively identify and type protein-coding variants throughout the genome. Coupled with growing databases that contain known variants, exome sequencing makes identification of genetic mutations and risk factors possible in families and samples that were deemed insufficiently informative for previous genetic studies. Not only does exome sequencing enable identification of mutations in families that were undetectable with linkage and positional cloning methods, but compared with these methods, it is also much quicker and cheaper. Use of exome sequencing has so far been successful in many rare diseases.

Where next?

Exome sequencing is being adopted widely and we can expect an abundance of mutation discovery, similar to the deluge of genome-wide-association findings reported over the past 5 years; it is expected to enable the discovery of not only rare causal variants, but also protein-coding risk variants. This method will have application in both the research and clinical arenas and sets the scene for the use of whole-genome sequencing.

Introduction

Progress towards a full resolution of the genetic basis of disease is being substantially aided by a fast-moving technological development, exome sequencing. This method promises to speed up discovery of the genetic causes of disease in both the research and the clinical setting.

The method of exome sequencing has been covered elsewhere.1 Although several methods exist, they all use a similar principle: reducing a genomic DNA sample to one that is enriched for the protein-coding regions of the genome (exons), followed by very high-throughput sequencing of the exon-enriched sample (figure). In short, this is a method for rapidly identifying protein-coding mutations, including missense, non-sense, splice site, and small deletion or insertion mutations.

Exome sequencing uses second-generation sequencing, which generates sequence data from hundreds of millions of short DNA fragments in parallel. The sequencing of input libraries is, to all intents and purposes, random; each of the fragments that happens to be in the DNA library and is applied to the sequencer has about an equal chance of being sequenced. Thus, directive sequencing of specific DNA fragments is determined by creating a DNA library solely consisting of, or enriched for, the DNA regions of interest. In the context of exome sequencing, this target selection is done with one of several enrichment products, each of which is intended to produce a DNA sample in which the content consists of the protein-coding and regulatory regions of the genome. This method has some limitations: first, coverage of regions of interest is not complete. In early experiments about 8% of the regions of interest were not captured by the enrichment strategy,2 and although the coverage has improved, it will probably never reach 100%; second, at present, this method is not useful for identifying repeat mutations (such as triplet repeats in spinocerebellar ataxia); and third, copy number variants are difficult to detect with exome sequencing. However, in view of the distribution of variants, exome-sequencing remains an efficient way to identify most mutations altering protein sequence in any single DNA sample. Although exome sequencing is quite new to the market, it has been rapidly adopted by the research community.

In this Rapid Review, I aim to review the likely use and success of exome sequencing in research and clinical settings.

Section snippets

Mendelian disorders

The primary successes for exome sequencing have been in finding mutations that cause rare, familial forms of disease. The strength of this approach lies in the comprehensive discovery of protein-coding variants throughout the genome. This means that DNA samples collected from small families and isolated affected individuals, which could not be used for mutation identification through traditional linkage and positional cloning, can now be used to discover mutations that cause disease.

Exome

Beyond Mendelian disorders

There is increasing interest in taking advantage of the power of exome sequencing in diseases that do not exhibit a simple Mendelian mode of transmission. Exome sequencing has particular potential in diseases caused by non-inherited or de-novo mutations. Typically, this potential can be achieved with complementary approaches: the first, and most simple, is to sequence a group of cases suspected to have disease-causing de-novo or non-inherited mutations and look for a gene that is commonly

Challenges and opportunities

Exome sequencing, and also genome sequencing, will probably have a substantial effect in the clinical setting, beyond the identification of genes that were previously not known to contain disease-causing mutations. In the context of genetic testing, instead of screening an inherently limited panel of genes for a specific set of diseases, why not just sequence the whole exome? This method opens the door to many possibilities—eg, rapid genetic diagnosis and screening, when known disease-causing

Where next?

Some hold the view that there is little purpose in finding new genetic causes of disease because we have made little progress in understanding the consequences of the mutations that we already know about. However, examples of gains in knowledge from the identification of genes suggest that this argument is ill-founded; for instance, the role of amyloid processing in Alzheimer's disease was indicated by the discovery of APP, PS1, and PS2 mutations.39 This view also does not take into account

Search strategy and selection criteria

I searched PubMed for articles published between Jan 1, 2000, and June 15, 2011, using the search term “exome”. Results were manually assessed to identify articles that were relevant to the topic of this Rapid Review. Only articles published in English were selected.

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