Exome Sequencing: Technical Methodology, Clinical Applications, and Comparative Analysis

Whole genome sequencing is now a rapid and accessible procedure.
The timeframe for sequencing an entire human genome is currently estimated at less than one week, with modern machines capable of completing the process in as little as 2 to 3 days.
The base cost for whole genome sequencing has decreased significantly, settling around the $\$1,000$ mark, or potentially slightly higher.
Despite the speed and lower cost of sequencing, whole genome analysis presents significant logistical challenges:
- Data magnitude: Sequencing generates terabytes of data.
- Bioinformatics requirements: Extensive computer analysis is required to map the sequence data back to the reference human genome to identify mutations.
- Technical expertise: Because of the sheer volume of data, processing cannot be done over standard internet interfaces; it must be performed in specialized environments like the Unix system.
- Specialized Labor: Experts known as bioinformaticians, who specialize in computer programming and genetic mapping, are required for the analysis.

A primary issue with whole genome sequencing is the interpretability of the data: over $95\%$ of the genome has a function that is currently unknown.
Even when mutations, insertions, or deletions are discovered in these non-coding regions, scientific understanding is often insufficient to interpret how they contribute to disease.
This limitation has led to a common school of thought favoring the sequencing of only the parts of the genome that are understood and used for interpreting disease: the coding gene exons.
The collection of all exonic sequences from the coding genes is referred to as the exome.
The exome represents less than $1\%$ of the total human genome.

Speed and Data Volume: Since it represents such a small fraction of the genome, exome sequencing is significantly faster and produces far less data.
Ease of Analysis: Exome sequence data is much easier to analyze. Even researchers who are not computer literate can analyze an exome sequence, whereas whole genome analysis requires advanced bioinformatic skills.
Economic Considerations:
- The sequencing cost for an exome is approximately $\$600$ , compared to roughly $\$1,000$ for a genome.
- The primary cost difference lies in the bioinformatics hours. Analyzing a whole genome may cost an additional $\$1,000$ in professional hours, whereas exome analysis is simple enough for many researchers to do themselves.
Philosophical Justification: There is a practical and philosophical argument against whole genome sequencing: why expend resources to sequence information that cannot yet be utilized or understood?

Process Steps:
1. Obtain patient DNA.
2. Create a DNA library for sequencing.
3. Capture Step: Instead of sequencing all genomic DNA, an extra step is inserted to "pull out" only the exonic sequences through hybridization.
4. The transcript identifies one common method for this capture as microarray hybridization, though several methods exist.
5. The targeted DNA is washed off the capture medium.
6. The specific DNA sequences obtained mapping to the exons are then sequenced.
7. The resulting sequence is mapped against the reference genome to filter for variants within the exonic regions.
By excluding intergenic regions, researchers avoid the complication of common polymorphisms that likely have no disease relevance, making the discovery of causative mutations more feasible.

The Clinical Problem: A family presented with three out of four children losing their sight in their $30s$ due to retinitis pigmentosa.
Genetic Heterogeneity: Retinitis pigmentosa is known to be caused by mutations in over $50$ different genes.
Initial Screening: Clinicians initially screened and sequenced all $50$ known candidate genes but found no mutations.
Exome Sequencing Success: After the failure of traditional screening, exome sequencing was performed on selected family members. The causative gene was identified in less than six months.
The Identified Gene: The mutation was found in the DHDDS gene.
- Population Context: The family was of Ashkenazi Jewish descent, a genetically isolated population known to have a higher incidence of certain autosomal recessive mutations.
- Biological Function: The DHDDS protein is responsible for adding sugar groups to rhodopsin, a light-sensitive protein.
Validation via Model Organism: To confirm the findings, researchers introduced the mutations into zebrafish. The fish subsequently began to lose their eyesight, recapitulating the human phenotype and confirming the genetic cause.
Implications: Identifying this new protein involved in blindness enhances the understanding of genetic sight loss and opens avenues for novel therapeutic approaches, such as DHDDS protein replacement therapy.

Research Growth: Publications involving exome sequencing are on an exponential trajectory. In $2010$ , there were fewer than $100$ publications; recently, that number has reached between $2,000$ and $3,000$ .
Clinical Integration: Exome sequencing is moving from purely research environments into basic medical diagnostic testing.
Wide-Ranging Applications: Recent literature (circa $2016$ ) highlights the use of whole exome sequencing in:
- Autism spectrum disorders.
- Cardiovascular malformations.
- Rare bone disorders.
- Ovarian carcinomas.
Genetic Basis of Non-Genetic Conditions: Research even suggests a genetic basis for how patients respond in Intensive Care Units (ICU) following trauma, such as car accidents, by looking at genomic profiles and gene expression.

Newborn Screening: There is significant interest and ongoing discussion regarding the ethics and implementation of exome sequencing for newborn and underage screening.
Notable Institutions Providing Clinical Exome Sequencing:
- Children's Mercy in Kansas: Operates a pediatric genomic medicine exome sequencing facility.
- Cincinnati Children's Hospital: Provides whole exome sequencing services for healthcare professionals and patients.
- Sick Kids in Toronto: A long-standing leader in using whole exome sequencing for pediatric diagnostic testing.
- Garvan Institute in Sydney, Australia: Developed "Genome One" to provide clinical whole genome sequencing.
While most Australian research institutes use this methodology for research, the Garvan Institute is noted for its direct clinical link to patient diagnostics.