Exome quencing (also known as targeted exome capture) is an efficient strategy to lectively quence the coding regions of the human genome to identify novel genes associated with rare and common disorders[1] . Routine whole genome quencing of large numbers of individuals is still not feasible partly due to the high cost associated with the technique. At prent, it is necessary to u an alternative approach, in which certain regions of the genome, such as the “exome”, are targeted, enriched and quenced, which requires ~5% as much quencing as a whole genome[2]. The “exome” reprents all the exons in the human genome (i.e., the
protein-coding region of the genome). Exons are short, functionally important quences of DNA which reprent the regions in genes that are translated into protein. In total there are about 180,000 exons found in the human genome. The protein coding regions constitute about 1% of the human genome which translates to about 30 megabas (Mb) in length[2]. It is estimated that the protein coding regions of the human genome constitute about 85% of the dia-causing mutations[3].
Exome Sequencing Workflow: Part 1.
Exome Sequencing Workflow: Part 2.
The robust approach to quencing the complete coding region (exome) has the potential to be clinically relevant in genetic diagnosis due to current understanding of functional conquences in quence variation [3]. The goal of this approach is to identify the functional variation that is responsible for both mendelian and common dias such as Miller syndrome and Alzheimer’s dia without the high costs associated with whole-genome quencing while maintaining high coverage in quence depth[3].
As an efficient strategy
Exome quencing is an efficient strategy to identify the rare causal variants of mendelian disorders over whole genome quencing due to few factors:
1.Positional cloning strategies have reduced power to successfully identify causal rare
variants [1]
2.The majority of genetic variants that underlie mendelian disorders disrupt protein-coding
quences [1]
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3. A large number or rare nonsynonymous substitutions are predicted to be deleterious [1]lorac
coroner4.Splice sites also reprent quences in which there is high functional variation [1]
The exome reprents an enriched portion of the genome that can be ud to arch for variants with large effect sizes [1]. Mendelian disorders
Rare dias affect less than 200,000 individuals in the United States and are of interest becau the identification of the genetic basis can provide knowledge about biological pathways and therapeu
tic targets. It is suspected that there are more than 7,000 rare mendelian dias which affect millions of people in the US[1]. The majority of mendelian dias studied to date are known to be caud by rare mutations that affect protein function. The majority of mutations that are known to cau mendelian disorders are located in protein-coding regions while non-coding regions on the other hand are likely to have weak or neutral effects.
biomarkerTo 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. The include sample size of affected individuals, reduced penetrance, locus heterogeneity, and alleles that impair reproductive fitness [1]. The factors make it difficult to map the traits by linkage analysis and they reduce the power of traditional positional cloning strategies to identify the variants. For both dominant and recessive traits finding an excess of independent mutations
in the same locus will provide evidence that a dia gene has been identified [3]. Exome quencing is a powerful technique to identify genes in rare mendelian disorders becau it requires only a small number of unrelated cas to identify a causal gene. Technological platforms
The technical platforms ud to carry out exome quencing are DNA microarrays and magnetic bea
d bad systems for the enrichment of the exome DNA and next-generation quencing technologies.
Target-enrichment strategies
Target-enrichment methods allow to lectively capture genomic regions of interest from a DNA sample prior to quencing. Several target-enrichment strategies have been developed.
PCR
Uniplex and Multiplex PCR.
PCR is one of the most widely ud enrichment strategies for over 20 years [4]. This approach is known to be uful in classical Sanger quencing becau a uniplex PCR ud to generate a single DNA quence is comparable in read length to a typical amplicon. Mutliplex PCR reactions wh
ich require veral 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 bad approach is highly effective, yet it is not feasible to target genomic regions that are veral megabas in size due to quantity of DNA required and cost. Molecular Inversion Probes (MIP)
Molecular Inversion Probes.
unusualThis is an enzymatic technique that targets the amplification of genomic regions by multiplexing bad on target circularization. Accurate genotypes can be achieved from massively parallel quencing using this method. This method is suggested to be uful 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 ts [4].
Hybrid capture
In-Solution Capture.
This technique involves hybridizing shotgun libraries of genomic DNA to target-specific quences on a microarray[4]. Roche NimbleGen was first to take this technology and adapt it for next-generation quencing. They developed the Sequence Capture Human Exome 2.1M Array to capture ~180,000 coding exons[3]. This method is both time-saving and cost-effective compared to PCR bad methods. The Agilent Capture Array and the comparative genomic hybridization array also other methods that can be ud for hybrid capture of target quences. Limitations in this technique include the need for expensive hardware as well as a relatively large amount of DNA [4].
火龙果的英文In-solution capture
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) lectively 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 quenced allowing for lective DNA quencing of genomic regions (e.g. exons) of interest. Several companies (e.g. FlexGen) offer custom pools of oligonucleotides or instruments to synthesize the oligopools in-hou. This method was developed to improve on the hybridization capture target-enrichment method. In solution capture as oppod to hybrid capture, there is an excess of probes to target regions of interest over the amount of template required [4]. The optimal target size is about 3.5 Mb in length and yields excellent quence coverage of the target regions. The preferred method is dependent on veral factors including; size (bp) of region of interest, demands for reads on target, equipment in hou, etc. [5]
Sequencing
There are veral quencing platforms available including the classical Sanger quencing. Other platforms include the Roche 454 quencer, the Illumina Genome Analyzer II and the Applied Biosys
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tems SOLiD, which have both been ud for exome quencing. Significance
A study published in September 2009 discusd a proof of concept experiment to determine if it was possible to identify causal genetic variants using exome quencing. They quenced four individuals with Freeman-Sheldon syndrome (FSS) (OMIM 193700), a rare autosomal dominant disorder known to be caud by a mutation in the gene MYH3[2]. Eight HapMap individuals were also quenced 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 quencing can be ud to identify causal variants of rare disorders[2]. This is the first reported study that ud exome quencing as an approach to identify an unknown causal gene for a rare mendelian disorder.
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英语大师hannahA cond report was conducted on exome quencing 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
