Exome sequencing (also known as targeted exome capture) is an efficient strategy to selectively sequence the coding regions of the genome as a cheaper but still effective alternative to whole genome sequencing. A common application is to identify novel mutations associated with rare and common disorders. [1] [2] Routine whole genome sequencing of large numbers of individuals is still not feasible mainly due to its high cost. At present, it is necessary to use an alternative approach, in which certain regions of the genome, such as the “exome”, are targeted, enriched and sequenced, which requires only ~5% of the sequencing a whole genome requires.[3] The “exome” represents all the exons in the human genome (i.e., the transcribed regions of the genome). Exons are short, functionally important sequences of DNA which represent the regions in genes that are translated into protein and untranslated region flanking them (UTR). UTRs are usually not included in exome studies. In total there are about 180,000 exons found in the human genome. These protein coding regions constitute about 1% of the human genome which translates to about 30 megabases (Mb) in length.[3] It is estimated that the protein coding regions of the human genome constitute about 85% of the disease-causing mutations.[4]
The robust approach to sequencing the complete coding region (exome) has the potential to be clinically relevant in genetic diagnosis due to current understanding of functional consequences in sequence variation.[4] The goal of this approach is to identify the functional variation that is responsible for both mendelian and common diseases such as Miller syndrome and Alzheimer’s disease without the high costs associated with whole-genome sequencing while maintaining high coverage in sequence depth.[4]
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Exome sequencing is an efficient strategy to identify these rare causal variants of mendelian disorders over whole genome sequencing due to few factors:
The exome represents an enriched portion of the genome that can be used to search for variants with large effect sizes.[2]
Rare diseases affect less than 200,000 individuals in the United States and are of interest because the identification of the genetic basis can provide knowledge about biological pathways and therapeutic targets. It is suspected that there are more than 7,000 rare mendelian diseases which affect millions of people in the US.[2] The majority of mendelian diseases studied to date are known to be caused by rare mutations that affect protein function. The majority of mutations that are known to cause mendelian disorders are located in protein-coding regions while non-coding regions on the other hand are likely to have weak or neutral effects.
To date, less than half of all rare monogenic disorders have been discovered. The identification of genetic variants for rare disorders is limited by a number of factors. These include sample size of affected individuals, reduced penetrance, locus heterogeneity, and alleles that impair reproductive fitness.[2] These factors make it difficult to map these traits by linkage analysis and they reduce the power of traditional positional cloning strategies to identify these variants. For both dominant and recessive traits finding an excess of independent mutations in the same locus will provide evidence that a disease gene has been identified.[4] Exome sequencing is a powerful technique to identify genes in rare mendelian disorders because it requires only a small number of unrelated cases to identify a causal gene.
The technical platforms used to carry out exome sequencing are (1) DNA microarrays and magnetic bead based systems for the enrichment of the exome DNA and (2) next-generation sequencing technologies.
Target-enrichment methods allow to selectively capture genomic regions of interest from a DNA sample prior to sequencing. Several target-enrichment strategies have been developed since the original description of the Direct Genomic Selection (DGS) method by the Lovett group in 2005.[1]
PCR is one of the most widely used enrichment strategies for over 20 years.[5] This approach is known to be useful in classical Sanger sequencing because a uniplex PCR used to generate a single DNA sequence is comparable in read length to a typical amplicon. Multiplex PCR reactions which require several primers are challenging although strategies to get around this have been developed. A limitation to this method is the size of the genomic target due to workload and quantity of DNA required. The PCR based approach is highly effective, yet it is not feasible to target genomic regions that are several megabases in size due to quantity of DNA required and cost.
This is an enzymatic technique that targets the amplification of genomic regions by multiplexing based on target circularization. Accurate genotypes can be achieved from massively parallel sequencing using this method. This method is suggested to be useful for small numbers of targets in a large number of samples. Major disadvantage of this method for target enrichment is the capture uniformity as well as the cost associated with covering large target sets.[5]
This technique involves hybridizing shotgun libraries of genomic DNA to target-specific sequences on a microarray.[5] Roche NimbleGen was first to take the original DGS technology [1] and adapt it for next-generation sequencing. They developed the Sequence Capture Human Exome 2.1M Array to capture ~180,000 coding exons.[4] This method is both time-saving and cost-effective compared to PCR based methods. The Agilent Capture Array and the comparative genomic hybridization array are other methods that can be used for hybrid capture of target sequences. Limitations in this technique include the need for expensive hardware as well as a relatively large amount of DNA.[5]
To capture genomic regions of interest using in-solution capture, a pool of custom oligonucleotides (probes) is synthesized and hybridized in solution to a fragmented genomic DNA sample. The probes (labeled with beads) selectively hybridize to the genomic regions of interest after which the beads (now including the DNA fragments of interest) can be pulled down and washed to clear excess material. The beads are then removed and the genomic fragments can be sequenced allowing for selective DNA sequencing of genomic regions (e.g. exons) of interest.
This method was developed to improve on the hybridization capture target-enrichment method. In solution capture as opposed to hybrid capture, there is an excess of probes to target regions of interest over the amount of template required.[5] The optimal target size is about 3.5 Mb in length and yields excellent sequence coverage of the target regions. The preferred method is dependent on several factors including; size (bp) of region of interest, demands for reads on target, equipment in house, etc.[6]
There are several sequencing platforms available including the classical Sanger sequencing. Other platforms include the Roche 454 sequencer, the Illumina Genome Analyzer II and the Applied Biosystems SOLiD, all of which have been used for exome sequencing.
A study published in September 2009 discussed a proof of concept experiment to determine if it was possible to identify causal genetic variants using exome sequencing. They sequenced four individuals with Freeman-Sheldon syndrome (FSS) (OMIM 193700), a rare autosomal dominant disorder known to be caused by a mutation in the gene MYH3.[3] Eight HapMap individuals were also sequenced to remove common variants in order to identify the causal gene for FSS. After exclusion of common variants, the authors were able to identify MYH3, which confirms that exome sequencing can be used to identify causal variants of rare disorders.[3] This was the first reported study that used exome sequencing as an approach to identify an unknown causal gene for a rare mendelian disorder.
Subsequently, another group reported successful clinical diagnosis of a suspected Bartter syndrome patient of Turkish origin.[4] Bartter syndrome is a renal salt-wasting disease. Exome sequencing revealed an unexpected well-conserved recessive mutation in a gene called SLC26A3 which is associated with congenital chloride diarrhea (CLD). This molecular diagnosis of CLD was confirmed by the referring clinician. This example provided proof of concept of the use of whole-exome sequencing as a clinical tool in evaluation of patients with undiagnosed genetic illnesses. This report is regarded as the first application of next generation sequencing technology for molecular diagnosis of a patient.
A second report was conducted on exome sequencing of individuals with a mendelian disorder known as Miller syndrome (MIM#263750), a rare disorder of autosomal recessive inheritance. Two siblings and two unrelated individuals with Miller syndrome were studied. They looked at variants that have the potential to be pathogenic such as non-synonymous mutations, splice acceptor and donor sites and short coding insertions or deletions.[2] Since Miller syndrome is a rare disorder, it is expected that the causal variant has not been previously identified. Previous exome sequencing studies of common single nucleotide polymorphisms (SNPs) in public SNP databases were used to further exclude candidate genes. After exclusion of these genes, the authors found mutations in DHODH that were shared among individuals with Miller syndrome. Each individual with Miller syndrome was a compound heterozygote for the DHODH mutations which were inherited as each parent of an affected individual was found to be a carrier.[2]
This was the first time exome sequencing was shown to identify a novel gene responsible for a rare mendelian disease. This exciting finding demonstrates that exome sequencing has the potential to locate causative genes in complex diseases, which previously has not been possible due to limitations in traditional methods. Targeted capture and massively parallel sequencing represents a cost-effective, reproducible and robust strategy with high sensitivity and specificity to detect variants causing protein-coding changes in individual human genomes.
There are multiple technologies available to undertake methods to identify causal genetic variants associated with disease. Each technology has its own technical, financial and throughput limitations. Microarrays, for example, require hybridization probes of known sequence and are therefore limited by probe design and thus prevent the identification of genetic changes that can be detected.[5] Massively parallel sequencing technologies used for exome sequencing, on the other hand, make it now possible to identify the cause of many unknown diseases by screening thousands of loci at once.[7] This technology addresses the present limitations of hybridization genotyping arrays and classical sequencing.
Although exome sequencing is an expensive method relative to other technologies (e.g., hybridization-based technologies) currently available, it is an efficient strategy to identify the genetic bases that underlie rare mendelian disorders. This approach has become increasingly practical with the falling cost and increased throughput of whole genome sequencing. Even by only sequencing the exomes of individuals, a large quantity of data and sequence information is generated which requires a significant amount of data analysis. Challenges associated with the analysis of this data include changes in programs used to align and assemble sequence reads.[5] Various sequence technologies also have different error rates and generate various read-lengths which can pose challenges in comparing results from different sequencing platforms.
Exome sequencing is only able to identify those variants found in the coding region of genes which affect protein function. It is not able to identify the structural and non-coding variants associated with the disease, which can be found using other methods such as whole genome sequencing.[3] There remains 99% of the human genome that is not covered using exome sequencing. Whole genome sequencing will eventually become a standard approach and allow us to gain a deeper understanding of genetic variation found in populations. Presently, this technique is not practical due to the high costs and time associated with sequencing large numbers of genomes. Exome sequencing allows sequencing of portions of the genome over at least 20 times as many samples compared to whole genome sequencing.[3] For translation of identified rare variants into the clinic, sample size and the ability to interpret the results to provide a clinical diagnosis indicates that with the current knowledge in genetics, exome sequencing may be the most valuable.[4]
The statistical analysis of the large quantity of data generated from sequencing approaches is a challenge. False positive and false negative findings are associated with genomic resequencing approaches and is a critical issue. A few strategies have been developed to improve the quality of exome data such as:
Rare recessive disorders would not have single nucleotide polymorphisms (SNPs) in public databases such dbSNP. More common recessive phenotypes may have disease-causing variants reported in dbSNP. For example, the most common cystic fibrosis variant has an allele frequency of about 3% in most populations. Screening out such variants might erroneously exclude such genes from consideration. Genes for recessive disorders are usually easier to identify than dominant disorders because the genes are less likely to have more than one rare nonsynonymous variant.[3] The system that screens common genetic variants relies on dbSNP which may not have accurate information about the variation of alleles. Using lists of common variation from a study exome or genome-wide sequenced individual would be more reliable. A challenge in this approach is that as the number of exomes sequenced increases, dbSNP will also increase in the number of uncommon variants. It will be necessary to develop thresholds to define the common variants that are unlikely to be associated with a disease phenotype.[7]
Genetic heterogeneity and population ethnicity are also major limitations as they may increase the number of false positive and false negative findings which will make the identification of candidate genes more difficult. Of course, it is possible to reduce the stringency of the thresholds in the presence of heterogeneity and ethnicity, however, it will reduce the power to detect variants as well.
New technologies in genomics have changed the way researchers approach both basic and translational research. With approaches such as exome sequencing, it is possible to significantly enhance the data generated from individual genomes which has put forth a series of questions on how to deal with the vast amount of information. Should the individuals in these studies be allowed to have access to their sequencing information? Is it possible to interpret these results for these individuals and are the identified genetic variants clinically relevant? This data can lead to unexpected findings and complicate clinical utility and patient benefit. This area of genomics still remains a challenge and researchers are looking into how to address these questions.[7]
On September 29th, 2011 Ambry Genetics added the Clinical Diagnostic Exome, making them the first CLIA-certified laboratory to offer exome sequencing along with medical interpretation for clinical diagnostic purposes[8]. The company states that results from exome sequencing will allow clinicians to diagnose affected patients with conditions that have eluded traditional diagnostic approaches.
Identification of the underlying disease gene mutation(s) can have major implications for diagnostic and therapeutic approaches, can guide prediction of disease natural history, and makes it possible to test at-risk family members. [2][3][9][10][11][12] There are many factors that make exome sequencing superior to single gene analysis including the ability to identify mutations in genes that were not tested due to an atypical clinical presentation [12] or the ability to identify clinical cases where mutations from different genes contribute to the different phenotypes in the same patient.[2]
The authors of the major peer-reviewed publications on exome sequencing clearly emphasize the clinical utility. Using exome sequencing to identify the underlying mutation for a patient with Bartter Syndrome and congenital chloride diarrhea, the authors state: “We can envision a future in which such information will become part of the routine clinical evaluation of patients with suspected genetic diseases in whom the diagnosis is uncertain… We anticipate that whole-exome sequencing will make broad contributions to understanding the genes and pathways that contribute to rare and common human diseases, as well as clinical practice”. [9] Bilgular’s group also used exome sequencing and identified the underlying mutation for a patient with severe brain malformations, stating “[These findings]highlight the use of whole exome sequencing to identify disease loci in settings in which traditional methods have proved challenging… Our results demonstrate that this technology will be particularly valuable for gene discovery in those conditions in which mapping has been confounded by locus heterogeneity and uncertainty about the boundaries of diagnostic classification, pointing to a bright future for its broad application to medicine.” [10] Likewise, whole exome sequencing was performed in a child with intractable inflammatory bowel disease. After dozens of uninformative test results, over 100 surgical procedures, and clinical consultations with physicians from around with world to little success, exome sequencing identified the underlying mutation. The mutation identification, and knowledge of the gene function, guided treatment which involved a bone marrow transplantation which cured the child of disease. In the seminal publication, the authors explain that the case “…demonstrates the power of exome sequencing to render a molecular diagnosis in an individual patient in the setting of a novel disease, after all standard diagnoses were exhausted, and illustrates how this technology can be used in a clinical setting”.[11]
In September 2011, the whole exome sequencing services geared toward individuals was announced by 23andMe. Individuals have had access to whole genome sequencing services for some time through companies like Illumina and Knome, but at a cost of several thousand dollars and often the services were geared toward researchers. The pilot program announced by 23andMe costs $999 and requires no physician signature, but provides only raw data without analysis.[13][14]