Next Generation Sequencing (NGS) is a sequencing technology characterized by massive parallel data production compared to conventional Sanger sequencing. Although the cost of sequencing continues to decrease with advances in NGS technology, target capture method still plays a key role for improving sequencing efficiency and cost-effectiveness in NGS experiment. Currently, there are two major target capture technologies: multiplex PCR and hybridization capture.
- Multiplex PCR
Multiplex PCR is a technique used to amplify multiple target DNA sequences simultaneously in a single PCR reaction. This method uses multiple sets of primers, each designed to target a specific region of interest. PCR amplification has advantages that the experimental method is very simple and does not require special reagents or equipment. However, amplification bias and off-target effects can frequently occur in multiplex PCR, so the size of the region that can be captured at one reaction is limited. And if there is a mutation at the primer binding site, the corresponding primer cannot bind and thus that specific region will not be amplified. In addition, since all DNA molecules amplified by the same primer pair have the same form, which makes the analysis challenging for removing PCR duplicates.
- Hybridization Capture
Hybridization capture is a technique to selectively capture and enrich target DNA sequences in a sample. This method uses biotinylated probes that are complementary to the target regions of interest. The probes hybridize to the target DNA, and the biotinylated probes are then captured and isolated using streptavidin-coated magnetic beads. Compared to PCR amplification, the experimental process may seem complicated as additional reagents and equipment are required. However, hybridization capture method can be beneficial because there is no limitation to increase the size of target region as there are no interference between capture probe sets. As the length of the probe is much longer than the PCR primer, it can still capture proper target even if a mutation occurs. In addition, since the shape of the DNA molecule is determined by random shearing included in the NGS library prep process, it is possible to remove PCR duplicates during data analysis process.
This is an example of NGS data generated by two target enrichment techniques.
Both multiplex PCR and hybridization capture are useful methods for targeted DNA sequencing. Multiplex PCR is a cost-effective and efficient way to target a small number of genes or regions, while hybridization capture is a powerful tool for targeting a large number of genes or regions with high specificity and minimal off-target effects. The choice between these two methods ultimately depends on your explicit research needs and the available resources.