What is Capture-Seq and How Does It Work?

Capture-Seq is a targeted sequencing method that focuses on specific regions of an organism’s genetic material. Instead of analyzing an entire genome or transcriptome, which can be resource-intensive, this technique uses probes to isolate predefined areas of interest. This selective recovery allows for a more detailed examination of these specific parts. The method is used for various research purposes, from identifying genetic variations to analyzing complex genomes.

Genetics 101: DNA, RNA, and Genes

Deoxyribonucleic acid (DNA) is a molecule composed of two chains that form a double helix, carrying the genetic instructions for all known organisms. These instructions are written in a code of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). The sequence of these bases along a DNA strand determines the specific instructions.

A gene is a specific sequence of DNA bases that provides instructions for making a protein or functional RNA molecule. These proteins perform a vast array of tasks within the cell, such as acting as enzymes or structural components. The complete set of DNA in an organism is called its genome.

Ribonucleic acid (RNA) is chemically similar to DNA and one of its primary roles is to act as a messenger. This messenger RNA (mRNA) carries instructions from DNA to the cell’s protein-making machinery. The process of creating an mRNA molecule from a DNA template is called transcription. The complete set of RNA transcripts in a cell is the transcriptome, providing a snapshot of active genes at a specific time.

The process of determining the precise order of bases in a DNA or RNA molecule is called sequencing. Sequencing allows researchers to read the genetic blueprint and identify variations or patterns associated with different traits or diseases. Capture-Seq is a specialized form of sequencing that focuses on reading pre-selected parts of the genome or transcriptome with high accuracy.

How Capture-Seq Targets Specific Genetic Information

Capture-Seq selectively isolates specific DNA or RNA sequences from a complex mixture of genetic material. This process relies on probes, which are short, single-stranded pieces of DNA or RNA designed to be complementary to the target sequences. The probes seek out and bind to their targets through a process called hybridization.

The process begins with preparing a sample by extracting DNA or RNA from cells. Long DNA strands are fragmented into smaller pieces. If RNA is the target, it is converted into a more stable form called complementary DNA (cDNA) using an enzyme called reverse transcriptase. This collection of fragmented DNA or cDNA is known as a library.

Once the library is prepared, it is mixed with the custom-designed probes. The mixture is heated to separate the double-stranded DNA or cDNA into single strands. This allows the probes to find and bind to their complementary targets as the mixture cools, a process which tags the desired sequences for capture.

After hybridization, the probe-bound sequences are physically separated from the rest of the genetic material, for example by using magnetic beads that bind to the probes. The unbound fragments are washed away, leaving a sample highly enriched for the target DNA or RNA sequences. This enriched library is then ready for sequencing.

Reading the Captured Code: The Sequencing Process

After the target regions are isolated, the sequencing process begins. This step uses Next-Generation Sequencing (NGS) to determine the precise order of nucleotide bases in the captured fragments. NGS platforms can sequence millions of fragments simultaneously, generating a massive amount of data in a short time.

Because the sample is enriched for specific regions, the sequencing effort is concentrated on these areas. This allows for “deep” sequencing, where each targeted base is read many times. This high coverage increases confidence in the results and allows for the detection of rare variants or transcripts that other methods might miss.

The output is a large collection of short sequence reads, which are aligned to a reference genome to determine their original location. Analyzing the aligned reads allows researchers to detect single nucleotide polymorphisms (SNPs), which are variations at a single DNA position. They can also identify insertions, deletions, and other structural variations.

When applied to RNA, the method provides a detailed picture of gene expression. The number of reads mapping to a gene is proportional to that gene’s transcript abundance in the original sample, allowing researchers to quantify expression levels. The high resolution of Capture-Seq is also well-suited for discovering novel exons, alternative splice isoforms, and unannotated transcripts.

Why Use Capture-Seq: Discoveries and Uses

Human Genetics

In human genetics, Capture-Seq is used to identify the genetic causes of inherited diseases. By targeting a panel of genes known to be associated with a condition, researchers can efficiently screen patients for disease-causing mutations. This approach is more cost-effective and provides higher quality data for the regions of interest compared to whole-genome sequencing.

Cancer Research

Capture-Seq is used to analyze the genetic makeup of tumors. Sequencing a panel of cancer-related genes helps researchers identify mutations driving tumor growth that may be targeted by specific therapies. This information guides treatment decisions and leads to more personalized cancer care. The method can also monitor a tumor’s evolution and detect the emergence of drug resistance.

Agriculture

In agriculture, the method is used for allele mining, which is the search for beneficial gene variants in crop plants to improve traits like yield, disease resistance, and nutritional value. It is also used for genetic fingerprinting, QTL mapping, and studying the complex genomes of polyploid species. These applications help accelerate breeding programs and contribute to global food security.

Ecology and Evolution

In ecology and evolution, Capture-Seq is used to study population genetic diversity and the relationships between species. It can track the spread of pathogens and the evolution of resistance genes. Because the method targets specific regions, it is a cost-effective way to obtain genetic data from a large number of samples.