In 2003, the Human Genome Project and Celera Corporation mapped the human genome. Yet today, even with the full genome available as a reference, finding disease-causing mutations in someone’s DNA is still harder than finding a needle in a haystack.
Exome sequencing may provide the solution.
All the protein-coding regions of the genome make up around 1%-2% of the total DNA sequence, and are known collectively as the “exome.” These regions are distributed throughout the genome, surrounded by other non-coding DNA regions. Exome regions are critically important for understanding the genetic causes of many diseases because changing even one base pair in the exome can potentially destroy the function of a protein. While the exome makes up only about 1% of the entire human genome, 85% of the genetic mutations known to cause disease can be found there.
Exome sequencing simplifies the search for disease-causing mutations by focusing strictly on the protein-coding regions. This dramatically reduces the amount of genomic DNA that needs to be sequenced to get meaningful information about a disease. In fact, by looking only at the exome, sequencing effectiveness can be increased by over 2,000%, reducing costs and wait times for clinical screening.
The process of enriching relevant regions of the genome for focused analysis by next-generation sequencing (NGS) is known as target enrichment. Here is how it’s done.
First, extract the genomic DNA from a sample and break it into small pieces. This pool of fragments is then prepared for next-generation sequencing. They are enzymatically blunted and a single adenine overhang is enzymatically ligated onto the strand. Onto this overhang, sequencing adapters (short DNA strands that initiate the sequencing reaction) are ligated.
Then the prepared fragment pool of genome fragments is heated to temperatures above 200°F, so that their double helix structure comes apart, creating a pool of single-strand DNA fragments. Add specially designed synthetic DNA strands (called probes) that match up with the DNA fragments you are interested in (the exome in this case), but not with the rest of the DNA fragments in the pool. By performing a reaction to attach only these small synthetic strands to magnetic beads, the target fragments of interest can be physically and selectively chosen from the pool of genetic material.
Target capture with magnetic beads.
Once the exome has been isolated through target enrichment, it can be sequenced just like any other genome, and compared to a reference genome to see if it contains any specific disease-causing variants.
To identify disease-causing variants, researchers need to see a large number of data points that consistently support their assessment, especially in cases of rare mutations. An important metric for this purpose is the “read depth” of the gene sequence, i.e. the number of sequenced fragments that map to a given nucleotide. The industry standard for read depth varies depending on the type of experiment being performed. For exome sequencing, 20x coverage is considered sufficient, meaning 20 sequenced fragments align with the nucleotide of interest. In searches for certain rare mutations, the industry standard can range as high as 10,000x. Target enrichment allows researchers to achieve their required read depth much more efficiently by focusing all their resources on the specific sections of the genome they wish to study.
Target enrichment is also proving its value in situations where DNA samples are scarce or of poor quality. For instance, HudsonAlpha Institute of Bioscience in Huntsville, Alabama, and Twist Bioscience used target enrichment to sequence tissues that were preserved with the DNA damaging compound Paraformaldehyde and then set in paraffin (FFPE). These samples, which are essentially embalmed human tissue, are often many decades old. Hospitals and research centers archive millions of them, so they could be a treasure trove of information about the genetic causes of diseases such as cancer and Alzheimer’s.
Shawn Levy, Ph.D., Director of the Genomic Services Laboratory at HudsonAlpha, explains the special challenges of sequencing these irreplaceable genetic samples: “A big problem is the quality of nucleic acid sample that comes out; it is really damaged during fixation.” Target enrichment solves this problem by focusing solely on the protein-coding regions in order to obtain the read depth and coverage required to draw correct conclusions from lower quality FFPE data.
Exome sequencing requires high-quality reagents and capture DNA to ensure accurate data that can be used in clinical research. An important consideration is the uniformity of the DNA capture. In other words, does the sequenced exome have sufficient data from every location, or are some parts of the exome insufficiently sequenced? Some parts of the exome are difficult to enrich, so uniformity can only be achieved by amplifying or overweighting the probes that target the hard-to-enrich sections. Twist Bioscience probes, for instance, are synthesized and amplified in a way that is highly uniform, so they capture all the regions of the exome equally well.
Twist Bioscience is unique in the enrichment market because it NGS QC tests its probes for quality before they ship to ensure that all probes are present at the correct concentrations to deliver uniform coverage and reproducible genome reads.
The Twist Human Core Exome Kit also offers the flexibility to select kit components in a modular fashion. Researchers can utilize the complete kit or easily add individual components to an established workflow. In addition, the target region of the Twist Human Core Exome Kit can be extended by researchers by adding application-specific custom content.
“We are providing the tools to prepare the DNA sample so that when it goes into NGS sequencing, the right parts are read effectively, the cost of the sample is as low as possible and the data you get is as complete as possible,” Leproust explained.
Dr. Levy is very positive about his experience with Twist’s Human Core Exome Kit. “The preparation of the library itself was very easy,” he says. “Many of the key points in the protocol were ‘addition only’ steps. The sample input requirement per experiment is significantly lower than other kits that we have worked with. This is really important when we’re dealing with the tiny, finite amount of FFPE preserved sample.”
By focusing on the regions of the genome where disease-causing mutations typically occur, exome sequencing can uncover important features of individual genomes quickly and efficiently. It can therefore deliver actionable insights sooner and at lower cost, while consuming fewer resources.
Twist Bioscience recently expanded its NGS solutions to include the Twist Human Core Exome Kit, a library preparation and target enrichment kit, able to run up to 96 samples simultaneously. Customer data utilizing the Twist Human Core Exome Kit and Twist Custom Panels was presented at the European Society of Human Genetics (ESHG) held in Milan on June 16-19, 2018.
This article is brought to you by Twist Bioscience, whose new Human Core Exome Kit can help improve the effectiveness of research screens. Learn more here about how Twist Bioscience is helping researchers focus on the 1%-2% of DNA sequences that matter most.